Polypeptides controlling phytate metabolism in plants

ABSTRACT

This invention relates to newly identified polynucleotides and polypeptides, variants and derivatives of same; methods for making the polynucleotides, polypeptides, variants, derivatives and antagonists. In particular the invention relates to polynucleotides and polypeptides of the phytate metabolic pathway.

[0001] This application is a divisional of co-pending U.S. applicationSer. No. 09/677,064 filed Aug. 29, 2000, which is a divisional of U.S.application Ser. No. 09/118,442 filed Jul. 17, 1998 now U.S. Pat. No.6,197,651, the disclosure of which is herein incorporated by reference.

FIELD OF THE INVENTION

[0002] The present invention relates to the field of animal nutrition.Specifically, the present invention relates to the identification anduse of genes encoding various enzymes involved in the metabolism ofphytate in plants and the use of these genes and mutants thereof toreduce the levels of phytate, and/or increase the levels of non-phytatephosphorus in food or feed.

BACKGROUND OF THE INVENTION

[0003] The role of phosphorus in animal nutrition is well recognized.Eighty percent of the phosphorus in the body of animals is found in theskeleton, providing structure to the animal. Twenty percent of thephosphorus in animals can be found in soft tissues, where it is aconstituent compound and therefore involved in a wide series ofbiochemical reactions. For example, phosphorus is required for thesynthesis and activity of DNA, RNA, phospholipids, and some B vitamins.

[0004] Though phosphorus is essential for healthy animals, it is alsorecognized that not all phosphorus in feed is bioavailable. Phytic acidsalts (i.e., phytates) are the major storage form of phosphorus inplants. See e.g., “Chemistry and Application of Phytic Acid: anOverview,” Phytic Acid: Chemistry and Application; Graf, Ed.; PilatusPress: Minneapolis, Minn., pp. 1-21; (1986). Phytates are the major formof phosphorus in seeds, typically representing from 50% to 80% of seedtotal phosphorus.

[0005] In corn and soybeans, for example, phytate represents about 60%to 80% of total phosphorus. When seed-based diets are consumed bynon-ruminants, the consumed phytic acid forms salts with severalnutritionally-important minerals in the intestinal tract. Excretion ofthese salts reduces the retention and utilization, i.e., bioavailabilityof the diet's phosphorus and mineral contents. Consequently, this canresult in mineral deficiencies in both humans and animals fed the aboveseed. See e.g., McCance et al., Biochem. J. 29:4269 (1935); Edman,Cereal Chem. 58:21 (1981).

[0006] Phytate, a large source of phosphorus, is not metabolized bymonogastric animals. Phytic acid, in fact, is considered to be ananti-nutritional factor because it reduces the bioavailability ofproteins and minerals by chelation; see e.g., Cheryan, “Phytic AcidInteractions in Food Systems,” CRC Crit. Rev. Food Sci. Nutr. 13:297-335(1980).

[0007] Phytate does not simply cause a reduction in nutrientavailability. The phytate-bound phosphorus in animal waste contributesto surface and ground water pollution. See e.g., Jongbloed et al.,Nether. J. Ag. Sci. 38:567 (1990).

[0008] Because the phytate content of seed has an impact on diet,phosphorus and mineral retention, and the environment, severalapproaches have been proposed to reduce this impact. Approaches includeremoving dietary phytate by post-harvest intervention and reducing seedphytate content genetically.

[0009] Post-harvest food processing methods that remove phytic acideither physically or via fermentation, are disclosed for example byIndumadhavi et al., Int. J. Food Sci. Tech. 27:221 (1992). Hydrolyzingphytic acid is a useful approach to increase the nutritional value ofmany plant foodstuffs. Phytases, as discussed more fully below, catalyzethe conversion of phytic acid to inositol and inorganic phosphate.Phytase-producing microorganisms include bacteria and yeasts. See e.g.Power et al., J. Bacteriol. 151:1102-1108 (1982); Segueilha et al.,Biotechnol. Lett. 15(4):399-404 (1993) and Nayini et al., Lebensm. Wiss.Technol. 17:24-26 (1984).

[0010] The use of phytases, phytic acid-specific phosphohydrolases,typically of microbial origin, as dietary supplements, is disclosed byNelson et al., J. Nutr. 101:1289 (1971). All currently knownpost-harvest technologies involve added procedures and expense in orderto circumvent problems associated with phytate.

[0011] The genetic approach involves developing crop germplasmpossessing heritable reductions in seed phytic acid. Heritablequantitative variation in seed phytic acid has been observed among linesof several crop species. See Raboy, In: Inositol Metabolism in Plants,Moore D. J., et al., (eds.) Alan R. Liss, New York, pp. 52-73; (1990).

[0012] However, this variation has been found to be highly andpositively correlated with variation in less desirable characteristics,therefore, breeding for reduced seed phytic acid using traditionalbreeding methods, could result in germplasm with undesirable correlatedcharacteristics. To date, there have been no reports of commerciallyacceptable low phytic acid corn germplasm produced by such an approach.

[0013] In genetically altering phytate, natural variability for phytateand free phosphorus has been examined. See Raboy, V. and D. B. DickinsonCrop Sci. 33:1300-1305 (1993),and Raboy, V. et al., Maydica35:383-390(1990). While some variability for phytic acid was observed,there was no corresponding change in non-phytate phosphorus. Inaddition, varietal variability represented only two percent of thevariation observed, whereas ninety-eight percent of the variation inphytate was attributed to environmental factors.

[0014] As mentioned above, studies of soybean and other crops haveindicated that altering genetic expression of phytate through recurrentselection breeding methods might have correlated undesirable results.See Raboy, V., D. B. Dickinson, and F. E. Below; Crop Sci. 24:431-434(1984); Raboy, V., F. E. Below, and D. B. Dickinson; J. Hered.80:311-315 (1989); Raboy, V., M. M. Noaman, G. A. Taylor, and S. G.Pickett; Crop Sci. 31:631-635; (1991).

[0015] While it has been proposed that a block in phytic acidaccumulation might be valuable in producing low phytic acid germplasmwithout the introduction of undesirable correlated responses, (See Raboyet al., Crop Sci. 33:1300 (1993)) employing such a traditional mutantselection approach has, in certain cases, revealed that homozygosity formutants associated with substantial reductions in phytic acid alsoproved to be lethal.

[0016] Myo-inositol is produced from glucose in three steps involvingthe enzymes hexokinase (EC 2.7.1.1), L-myo-inositol 1-phosphate synthase(EC 5.5.1.4) and L-myo-inositol 1-phosphate phosphatase (EC 3.1.3.25).The biosynthetic route leading to phytate is complex and not completelyunderstood. Without wishing to be bound by any particular theory of theformation of phytate, it is believed that the synthesis may be mediatedby a series of one or more ADP-phosphotransferases, ATP-dependentkinases and isomerases. A number of intermediates have been isolatedincluding for example 2 and 3 monophosphates, 1,3 and 2,6 di-phosphates,1,3,5 and 2,5,6 triphosphates, 1,3,5,6 and 2,3,5,6 tetra-phosphates, and1,2,4,5,6 and 1,2,3,4,6 penta-phosphates. Several futile cycles ofdephosphorylation and rephosphorylation of the P₅ and P6 forms have beenreported as well as a cycle involvingG6P→myoinositiol-1-phosphate→myo-inositol; the last step beingcompletely reversible, indicating that control of metabolic flux throughthis pathway may be important. This invention differs from the foregoingapproaches in that it provides tools and reagents that allows theskilled artisan, by the application of, inter alia, transgenicmethodologies to influence the metabolic flux in respect to the phyticacid pathway. This influence may be either anabolic or catabolic, bywhich is meant the influence may act to decrease the flow resulting fromthe biosynthesis of phytic acid and/or increase the degradation (i.e.,catabolism of phytic acid). A combination of both approaches is alsocontemplated by this invention.

[0017] As mentioned above, once formed phytate may be dephosphorylatedby phosphohydrolases, particularly 3-phytases typically found inmicroorganisms and 6-phytases the dominant form in plants. After theinitial event, both enzymes are capable of successive dephosphorylationof phytate to free inositol.

[0018] Accordingly, there have also been reports that plants can betransformed with constructs comprising a gene encoding phytase. See Penet al., PCT Publication WO 91/14782, incorporated herein in its entiretyby reference. Transgenic seed or plant tissues expressing phytases canthen be used as dietary supplements. However, this application has notbeen done to reduce seed phytic acid.

[0019] Based on the foregoing, there exists the need to improve thenutritional content of plants, particularly corn and soybean byincreasing non-phytate phosphorus and reducing seed phytate with noother obvious or substantial adverse effects.

SUMMARY OF THE INVENTION

[0020] It is therefore an object of the present invention to provideplants, particularly transgenic corn, which has enhanced levels ofnon-phytate phosphorus without corresponding detrimental effects.

[0021] It is a further object of the present invention to provideplants, particularly transgenic corn which have reduced levels ofphosphorus in the form of phytate without corresponding detrimentaleffects.

[0022] It is a further object of the present invention to providetransgenic plant lines with dominant, heritable phenotypes which areuseful in breeding programs designed to produce commercial products withimproved phosphorus availability and reduced phytate.

[0023] It is a further object of the present invention to improve animalperformance by feeding animals plants and parts thereof particularlyseeds with enhanced nutritional value.

[0024] It is a further object of the present invention to provide plantseeds, particularly corn seeds and resulting meal, that result in lessenvironmental contamination, when excreted, than do currently usedseeds.

[0025] These and other objects of the invention will become readilyapparent from the ensuing description.

[0026] An isolated polynucleotide is provided comprising a memberselected from the group consisting of:

[0027] (a) a polynucleotide encoding a polypeptide comprising SEQ IDNOS: 2, 6, 11, 17 or complement thereof;

[0028] (b) a polynucleotide of at least 25 nucleotides in length whichselectively hybridizes under stringent conditions to a polynucleotide ofSEQ ID NOS: 1, 5, 7, 10, 14, 15, 16 or a complement thereof, wherein thehybridization conditions include a wash step in 0.1× SSC at 60° C.;

[0029] (c) a polynucleotide having a sequence of a nucleic acidamplified from a Zea mays nucleic acid library using the primers of SEQID NOS: 3-4, 8-9, 12-13, or 18-19;

[0030] (d) a polynucleotide having at least 75% sequence identity to SEQID NO: 1, at least 60% sequence identity to SEQ ID NO: 5, at least 80%sequence identity to SEQ ID NO: 10, or at least 70% sequence identity toSEQ ID NO: 16, wherein the % sequence identity is based on the entirecoding region and is determined by the GAP program where the gapcreation penalty=50 and the gap extension penalty=3; and

[0031] (e) a polynucleotide comprising at least 20 contiguous bases ofthe polynucleotide of (a) through (c), or complement thereof.

[0032] According to the present invention, polypeptides that have beenidentified as novel phytate biosynthetic enzymes are provided.

[0033] An isolated polypeptide is provided comprising an amino acidsequence which has at least 80% sequence identity to SEQ ID NO: 2, atleast 35% sequence identity to SEQ ID NO: 6, at least 90% sequenceidentity to SEQ ID NO: 11 or at least 80% sequence identity to SEQ IDNO: 17, wherein the % sequence identity is based on the entire sequenceand is determined by the GAP program where the gap creation penalty=12and the gap extension penalty=4.

[0034] It is a further object of the invention, moreover, to providepolynucleotides that encode maize phytate biosynthetic enzymes,particularly polynucleotides that encode phosphatidylinositol 3-kinase,myo-inositol monophosphatase-3, myo-inositol 1,3,4-triphosphate 5/6kinase and myo-inositol 1-phosphate synthase.

[0035] In a particularly preferred embodiment of this aspect of theinvention the polynucleotide comprises the regions encodingphosphatidylinositol 3-kinase, myo-inositol monophosphatase-3,myo-inositol 1,3,4-triphosphate 5/6 kinase and myo-inositol 1-phosphatesynthase.

[0036] In another particularly preferred embodiment of the presentinvention polypeptides are isolated from Zea mays.

[0037] In accordance with this aspect of the present invention there isprovided a polynucleotide of at least 25 nucleotides in length whichselectively hybridizes under stringent conditions to the polynucleotidesset out below, or a complement thereof. As used herein, “stringentconditions” means the hybridization conditions include a wash step in0.1× SSC at 60° C.

[0038] In accordance with this aspect of the present invention there isprovided a polynucleotide having a sequence of a nucleic acid amplifiedfrom a Zea mays nucleic acid library using the primers set out in thesequences below.

[0039] In accordance with this aspect of the invention there areprovided isolated nucleic acid molecules encoding phytate biosyntheticenzymes, particularly those from Zea mays, mRNAs, cDNAs, genomic DNAsand, in further embodiments of this aspect of the invention,biologically, useful variants, analogs or derivatives thereof, orfragments thereof, including fragments of the variants, analogs andderivatives.

[0040] Other embodiments of the invention are naturally occurringallelic variants of the nucleic acid molecules in the sequences providedwhich encode phytate biosynthetic enzymes.

[0041] In accordance with another aspect of the invention there areprovided novel polypeptides which comprise phytate biosynthetic enzymesof maize origin as well as biologically, or diagnostically usefulfragments thereof, as well as variants, derivatives and analogs of theforegoing and fragments thereof.

[0042] It also is an object of the invention to provide phytatebiosynthetic polypeptides, particularly phosphatidylinositol 3-kinase,myo-inositol monophosphatase-3, myo-inositol 1,3,4-triphosphate 5/6kinase or myo-inositol 1-phosphate synthase polypeptide, that may beemployed for modulation of phytic acid synthesis.

[0043] In accordance with yet a further aspect of the present invention,there is provided the use of a polypeptide of the invention, orparticular fragments thereof.

[0044] It is another object of the invention to provide a process forproducing the polypeptides, polypeptide fragments, variants andderivatives, fragments of the variants and derivatives, and analogs ofthe foregoing.

[0045] In a preferred embodiment of this aspect of the invention thereare provided methods for producing the polypeptides comprising culturinghost cells having expressibly incorporated therein a polynucleotideunder conditions for expression of phytate biosynthetic enzymes in thehost and then recovering the expressed polypeptide.

[0046] In accordance with another object of the invention there areprovided products, compositions, processes and methods that utilize theaforementioned polypeptides and polynucleotides, for purposes includingresearch, biological, and agricultural.

[0047] In accordance with yet another aspect of the present invention,there are provided inhibitors to such polypeptides, useful formodulating the activity and/or expression of the polypeptides. Inparticular, there are provided antibodies against such polypeptides.

[0048] In accordance with certain embodiments of the invention there areprobes that hybridize to phytate biosynthetic enzyme polynucleotidesequences useful as molecular markers in breeding programs.

[0049] In certain additional preferred embodiments of this aspect of theinvention there are provided antibodies against the phytate biosyntheticenzymes. In certain particularly preferred embodiments in this regard,the antibodies are selective for the entire class the phytatebiosynthetic enzymes, irrespective of species of origin as well asspecies-specific antibodies, such as antibodies capable of specificimmune reactivity with for example, Zea mays phytate biosyntheticenzymes.

[0050] In accordance with yet another aspect of the present invention,there are provided phytate enzyme antagonists. Among preferredantagonists are those which bind to phytate biosynthetic enzymes so asto inhibit the binding of binding molecules or to stabilize the complexformed between the phytate biosynthetic enzyme and the binding moleculeto prevent further biological activity arising from the phytatebiosynthetic enzyme. Also among preferred antagonists are molecules thatbind to or interact with phytate biosynthetic enzymes so as to inhibitone or more effects of a particular phytate biosynthetic enzyme or whichprevent expression of the enzyme and which also preferably result in alowering of phytic acid accumulation.

[0051] Other objects, features, advantages and aspects of the presentinvention will become apparent to those of skill from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating preferredembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0052] This invention relates, in part, to newly identifiedpolynucleotides and polypeptides; variants and derivatives of thesepolynucleotides and polypeptides; processes for making thesepolynucleotides and these polypeptides, and their variants andderivatives and antagonists of the polypeptides; and uses of thesepolynucleotides, polypeptides, variants, derivatives and antagonists. Inparticular, in these and in other regards, the invention relates topolynucleotides and polypeptides of the phytate metabolic pathway, mostparticularly with the enzymes phosphatidylinositol 3-kinase,myo-inositol monophosphatase-3, myo-inositol 1,3,4-triphosphate 5/6kinase and myo-inositol 1-phosphate synthase and genes encoding same.

[0053] Glossary

[0054] The following illustrative explanations are provided tofacilitate understanding of certain terms used frequently herein,particularly in the Examples. The explanations are provided as aconvenience and are not limitative of the invention.

[0055] PHYTATE BIOSYNTHETIC ENZYME-BINDING MOLECULE, as used herein,refers to molecules or ions which bind or interact specifically withphytate biosynthetic enzyme polypeptides or polynucleotides of thepresent invention, including, for example enzyme substrates, cellmembrane components and classical receptors. Binding betweenpolypeptides of the invention and such molecules, including binding orinteraction molecules may be exclusive to polypeptides of the invention,which is preferred, or it may be highly specific for polypeptides of theinvention, which is also preferred, or it may be highly specific to agroup of proteins that includes polypeptides of the invention, which ispreferred, or it may be specific to several groups of proteins at leastone of which includes a polypeptide of the invention. Binding moleculesalso include antibodies and antibody-derived reagents that bindspecifically to polypeptides of the invention.

[0056] GENETIC ELEMENT, as used herein, generally means a polynucleotidecomprising a region that encodes a polypeptide or a polynucleotideregion that regulates replication, transcription or translation or otherprocesses important to expression of the polypeptide in a host cell, ora polynucleotide comprising both a region that encodes a polypeptide anda region operably linked thereto that regulates expression. Geneticelements may be comprised within a vector that replicates as an episomalelement; that is, as a molecule physically independent of the host cellgenome. They may be comprised within plasmids. Genetic elements also maybe comprised within a host cell genome; not in their natural state but,rather, following manipulation such as isolation, cloning andintroduction into a host cell in the form of purified DNA or in avector, among others.

[0057] HOST CELL, as used herein, is a cell which has been transformedor transfected, or is capable of transformation or transfection by anexogenous polynucleotide sequence. Exogenous polynucleotide sequence isdefined to mean a sequence not naturally in the cell. This includestransformation to incorporate additional copies of an endogenouspolynucleotide.

[0058] IDENTITY and SIMILARITY, as used herein, and as known in the art,are relationships between two polypeptide sequences or twopolynucleotide sequences, as determined by comparing the sequences. Inthe art, identity also means the degree of sequence relatedness betweentwo polypeptide or two polynucleotide sequences as determined by thematch between two strings of such sequences. Both identity andsimilarity can be readily calculated (Computational Molecular Biology,Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991). Methods commonly employed to determineidentity or similarity between two sequences include, but are notlimited to those disclosed in Carillo, H., and Lipman, D., SIAM J.Applied Math. 48:1073 (1988). Preferred methods to determine identityare designed to give the largest match between the two sequences tested.Methods to determine identity and similarity are codified in computerprograms. Typical computer program methods to determine identity andsimilarity between two sequences include, GCG program package (Devereux,J., et al., Nucleic Acids Research 12(1):387 (1984)), BLASTP, BLASTN,FASTA and TFASTA (Atschul, S. F. et al., J. Mol. Biol. 215:403 (1990)).

[0059] For purposes of defining the present invention, the Gap programis used. The algorithm used for the Gap program is that of Needleman andWunsch (J. Mol. Biol. 48:443-453 [1970]). The parameters used are asfollows: for nucleotide comparisons the gap creation penalty=50, gapextension penalty=3; for amino acid comparisons the gap creationpenalty=12, the gap extension penalty=4.

[0060] ISOLATED, as used herein, means altered “by the hand of man” fromits natural state; i.e., that, if it occurs in nature, it has beenchanged or removed from its original environment, or both. For example,a naturally occurring polynucleotide or a polypeptide naturally presentin a living organism in its natural state is not “isolated,” but thesame polynucleotide or polypeptide separated from the coexistingmaterials of its natural state is “isolated”, as the term is employedherein. For example, with respect to polynucleotides, the term isolatedmeans that it is separated from the chromosome and cell in which itnaturally occurs. As part of or following isolation, suchpolynucleotides can be joined to other polynucleotides, such as DNAs,for mutagenesis, to form fusion proteins, and for propagation orexpression in a host, for instance. The isolated polynucleotides, aloneor joined to other polynucleotides such as vectors, can be introducedinto host cells, in culture or in whole organisms. Introduced into hostcells in culture or in whole organisms, such DNAs still would beisolated, as the term is used herein, because they would not be in theirnaturally occurring form or environment. Similarly, the polynucleotidesand polypeptides may occur in a composition, such as media formulations,solutions for introduction of polynucleotides or polypeptides, forexample, into cells, compositions or solutions for chemical or enzymaticreactions, for instance, which are not naturally occurring compositions,and, therein remain isolated polynucleotides or polypeptides within themeaning of that term as it is employed herein.

[0061] LIGATION, as used herein, refers to the process of formingphosphodiester bonds between two or more polynucleotides, which mostoften are double stranded DNAs. Techniques for ligation are well knownto the art and protocols for ligation are described in standardlaboratory manuals and references, such as, for instance, Sambrooketal., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and Maniatis et al.,pg. 146, as cited below.

[0062] OLIGONUCLEOTIDE(S), as used herein, refers to shortpolynucleotides. Often the term refers to single-strandeddeoxyribonucleotides, but it can refer as well to single- ordouble-stranded ribonucleotides, RNA:DNA hybrids and double-strandedDNAs, among others. Oligonucleotides, such as single-stranded DNA probeoligonucleotides, often are synthesized by chemical methods, such asthose implemented on automated oligonucleotide synthesizers. However,oligonucleotides can be made by a variety of other methods, including invitro recombinant DNA-mediated techniques and by expression of DNAs incells and organisms. Initially, chemically synthesized DNAs typicallyare obtained without a 5′ phosphate. The 5′ ends of sucholigonucleotides are not substrates for phosphodiester bond formation byligation reactions that employ DNA ligases typically used to formrecombinant DNA molecules. Where ligation of such oligonucleotides isdesired, a phosphate can be added by standard techniques, such as thosethat employ a kinase and ATP. The 3′ end of a chemically synthesizedoligonucleotide generally has a free hydroxyl group and, in the presenceof a ligase, such as T4 DNA ligase, readily will form a phosphodiesterbond with a 5′ phosphate of another polynucleotide, such as anotheroligonucleotide. As is well known, this reaction can be preventedselectively, where desired, by removing the 5′ phosphates of the otherpolynucleotide(s) prior to ligation.

[0063] PLANT, as used herein, includes, but is not limited to plantcells, plant tissue and plant seeds.

[0064] PLASMIDS, as used herein, generally are designated herein by alower case p preceded and/or followed by capital letters and/or numbers,in accordance with standard naming conventions that are familiar tothose of skill in the art. Starting plasmids disclosed herein are eithercommercially available, publicly available, or can be constructed fromavailable plasmids by routine application of well known, publishedprocedures. Many plasmids and other cloning and expression vectors thatcan be used in accordance with the present invention are well known andreadily available to those of skill in the art. Moreover, those of skillreadily may construct any number of other plasmids suitable for use inthe invention. The properties, construction and use of such plasmids, aswell as other vectors, in the present invention will be readily apparentto those of skill from the present disclosure.

[0065] POLYNUCLEOTIDE(S),as used herein, generally refers to anypolyribonucleotide or polydeoxribonucleotide, which may be unmodifiedRNA or DNA or modified RNA or DNA. Thus, for instance, polynucleotidesas used herein refers to, among others, single-and double-stranded DNA,DNA that is a mixture of single- and double-stranded regions or single-,double- and triple-stranded regions, single- and double-stranded RNA,and RNA that is mixture of single- and double-stranded regions, hybridmolecules comprising DNA and RNA that may be single-stranded or, moretypically, double-stranded, or triple-stranded, or a mixture of single-and double-stranded regions. In addition, polynucleotide as used hereinrefers to triple-stranded regions comprising RNA or DNA or both RNA andDNA. The strands in such regions may be from the same molecule or fromdifferent molecules. The regions may include all of one or more of themolecules, but more typically involve only a region of some of themolecules. One of the molecules of a triple-helical region often is anoligonucleotide. As used herein, the term polynucleotide includes DNAsor RNAs as described above that contain one or more modified bases.Thus, DNAs or RNAs with backbones modified for stability or for otherreasons are “polynucleotides” as that term is intended herein. Moreover,DNAs or RNAs comprising unusual bases, such as inosine, or modifiedbases, such as tritylated bases, to name just two examples, arepolynucleotides as the term is used herein. It will be appreciated thata great variety of modifications have been made to DNA and RNA thatserve many useful purposes known to those of skill in the art. The termpolynucleotide as it is employed herein embraces such chemically,enzymatically or metabolically modified forms of polynucleotides, aswell as the chemical forms of DNA and RNA characteristic of viruses andcells, including inter alia, simple and complex cells.

[0066] POLYPEPTIDES, as used herein, includes all polypeptides asdescribed below. The basic structure of polypeptides is well known andhas been described in innumerable textbooks and other publications inthe art. In this context, the term is used herein to refer to anypeptide or protein comprising two or more amino acids joined to eachother in a linear chain by peptide bonds. As used herein, the termrefers to both short chains, which also commonly are referred to in theart as peptides, oligopeptides and oligomers, for example, and to longerchains, which generally are referred to in the art as proteins, of whichthere are many types. It will be appreciated that polypeptides oftencontain amino acids other than the 20 amino acids commonly referred toas the 20 naturally occurring amino acids, and that many amino acids,including the terminal amino acids, may be modified in a givenpolypeptide, either by natural processes, such as processing and otherpost-translational modifications, but also by chemical modificationtechniques which are well known to the art. Even the commonmodifications that occur naturally in polypeptides are too numerous tolist exhaustively here, but they are well described in basic texts andin more detailed monographs, as well as in a voluminous researchliterature, and they are well known to those of skill in the art. Amongthe known modifications which may be present in polypeptides of thepresent are, to name an illustrative few, acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphatidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cystine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,proteolytic processing, phosphorylation, prenylation, racemization,selenoylation, sulfation, transfer-RNA mediated addition of amino acidsto proteins such as arginylation, and ubiquitination. Such modificationsare well known to those of skill and have been described in great detailin the scientific literature. Several particularly common modifications,glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation, forinstance, are described in most basic texts, such as, for instancePROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York (1993). Many detailed reviews areavailable on this subject, such as, for example, those provided by Wold,F., Posttranslational Protein Modifications: Perspectives and Prospects,pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C.Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth.Enzymol. 182:626-646 (1990) and Rattan et al., Protein Synthesis:Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci.663:48-62 (1992). It will be appreciated, as is well known and as notedabove, that polypeptides are not always entirely linear. For instance,polypeptides may be branched as a result of ubiquitination, and they maybe circular, with or without branching, generally as a result ofposttranslation events, including natural processing event and eventsbrought about by human manipulation which do not occur naturally.Circular, branched and branched circular polypeptides may be synthesizedby non-translation natural process and by entirely synthetic methods, aswell. Modifications can occur anywhere in a polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. In fact, blockage of the amino or carboxyl group in apolypeptide, or both, by a covalent modification, is common in naturallyoccurring and synthetic polypeptides and such modifications may bepresent in polypeptides of the present invention, as well. For instance,the amino terminal residue of polypeptides made in E. coli or othercells, prior to proteolytic processing, almost invariably will beN-formylmethionine. During post-translational modification of thepeptide, a methionine residue at the NH₂-terminus may be deleted.Accordingly, this invention contemplates the use of both themethionine-containing and the methionine-less amino terminal variants ofthe protein of the invention. The modifications that occur in apolypeptide often will be a function of how it is made. For polypeptidesmade by expressing a cloned gene in a host, for instance, the nature andextent of the modifications in large part will be determined by the hostcell post-translational modification capacity and the modificationsignals present in the polypeptide amino acid sequence. For instance, asis well known, glycosylation often does not occur in bacterial hostssuch as, for example, E. coli. Accordingly, when glycosylation isdesired, a polypeptide should be expressed in a glycosylating host,generally a eukaryotic cell. Similar considerations apply to othermodifications. It will be appreciated that the same type of modificationmay be present in the same or varying degree at several sites in a givenpolypeptide. Also, a given polypeptide may contain many types ofmodifications. In general, as used herein, the term polypeptideencompasses all such modifications, particularly those that are presentin polypeptides synthesized by expressing a polynucleotide in a hostcell.

[0067] TRANSFORMATION, as used herein, is the process by which a cell is“transformed” by exogenous DNA when such exogenous DNA has beenintroduced inside the cell membrane. Exogenous DNA may or may not beintegrated (covalently linked) into chromosomal DNA making up the genomeof the cell. In prokaryotes and yeasts, for example, the exogenous DNAmay be maintained on an episomal element, such as a plasmid. Withrespect to higher eukaryotic cells, a stably transformed or transfectedcell is one in which the exogenous DNA has become integrated into thechromosome so that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theeukaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

[0068] VARIANT(S), as used herein, of polynucleotides or polypeptides,as the term is used herein, are polynucleotides or polypeptides thatdiffer from a reference polynucleotide or polypeptide, respectively.Variants in this sense are described below and elsewhere in the presentdisclosure in greater detail. With reference to polynucleotides,generally, differences are limited such that the nucleotide sequences ofthe reference and the variant are closely similar overall and, in manyregions, identical. As noted below, changes in the nucleotide sequenceof the variant may be silent. That is, they may not alter the aminoacids encoded by the polynucleotide. Where alterations are limited tosilent changes of this type, a variant will encode a polypeptide withthe same amino acid sequence as the reference. Also as noted below,changes in the nucleotide sequence of the variant may alter the aminoacid sequence of a polypeptide encoded by the reference polynucleotide.Such nucleotide changes may result in amino acid substitutions,additions, deletions, fusions and truncations in the polypeptide encodedby the reference sequence, as discussed below. With reference topolypeptides generally, differences are limited so that the sequences ofthe reference and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more substitutions, additions, deletions,fusions and truncations, which may be present in any combination.

[0069] GERMPLASM, as used herein, means a set of genetic entities whichmay be used in a conventional breeding program to develop new plantvarieties.

[0070] HIGH PHOSPHOROUS TRANSGENIC, as used herein, means an entitywhich, as a result of recombinant genetic manipulation, produces seedwith a heritable decrease in phytic acid percentage and/or increase innon-phytate phosphorous percentage.

[0071] PHYTIC ACID, as used herein, means myo-inositol tetraphosphoricacid, myo-inositol pentaphosphoric acid and myo-inositol hexaphosphoricacid. As a salt with cations, phytic acid is “phytate”.

[0072] NON-PHYTATE PHOSPHOROUS, as used herein, means total phosphorusminus phytate phosphorous.

[0073] NON-RUMINANT ANIMAL means an animal with a simple stomach dividedinto the esophageal, cardia, fundus and pylorus regions. A non-ruminantanimal additionally implies a species of animal without a functionalrumen. A rumen is a section of the digestive system where feedstuff/foodis soaked and subjected to digestion by micro-organisms before passingon through the digestive tract. This phenomenon does not occur in anon-ruminant animal. The term non-ruminant animal includes but is notlimited to humans, swine, poultry, cats and dogs.

[0074] As mentioned above, the present invention relates to novel phyticacid metabolic polypeptides and polynucleotides encoding same, amongother things, as described in greater detail below. Among thepolypeptides particularly useful for the practice of this inventioninclude but are not limited to D-myo-inositol-3-phosphate synthase,myo-inositol 1-phosphate synthase (otherwise referred to as INO1),phosphatidylinositol-4-phosphate-5-kinase, signaling inositolpolyphosphate-5-phosphatase (SIP-110), myo-inositol monophosphatase-3,myo-inositol 1,3,4 triphosphate 5/6 kinase, 1 D-myo-inositoltrisphosphate 3-kinase B, myo-inositol monophosphatase-1, inositolpolyphosphate 5-phosphatase, 1 D-myo-inositol trisphosphate 3-kinase,phosphatidylinositol 3-kinase, phosphatidylinositol 4-kinase,phosphatidylinositol synthase, phosphatidylinositol transfer protein,phosphatidylinositol 4,5-bisphosphate 5-phosphatase, myo-inositoltransporter, phosphatidylinositol-specific phospholipase C and maizephytase.

[0075] The nucleic acids and fragments thereof encoding theabove-mentioned enzymes are useful to generate enzyme deficienttransgenics. For example, a single gene or gene fragment (orcombinations of several genes) may be incorporated into an appropriateexpression cassette (using for example the globulin-1 promoter forembryo-preferred expression or the native promoter associated with theenzyme encoding gene) and transformed into corn along with anappropriate selectable marker (such as the herbicide PAT) in such amanner as to silence the expression of the endogenous genes.

[0076] Relevant literature describing the application ofhomology-dependent gene silencing include: Jorgensen, Trends Biotechnol8 (12):340-344 (1990); Flavell, Proc. Nat'l. Acad. Sci. (USA)91:3490-3496 (1994); Finnegan et al., Bio/Technology 12: 883-888 (1994);Neuhuberet al., Mol. Gen. Genet. 244:230-241 (1994). Alternatively,another approach to gene silencing can be with the use of antisensetechnology (Rothstein et al. in Osf. Surv. Plant Mol. Cell. Biol.6:221-246 (1989).

[0077] In particular, the invention relates to polypeptides andpolynucleotides of novel phytate biosynthetic enzyme genes. Theinvention relates especially to Zea mays phytate biosynthetic enzymeshaving the nucleotide and amino acid sequences set out belowrespectively.

[0078] Polynucleotides

[0079] In accordance with one aspect of the present invention, there areprovided isolated polynucleotides which encode the phytate biosyntheticenzymes having the deduced amino acid sequence below.

[0080] Using the information provided herein, such as the polynucleotidesequences set out below, a polynucleotide of the present inventionencoding phytate biosynthetic enzyme polypeptides may be obtained usingstandard cloning and screening procedures. To obtain the polynucleotideencoding the protein using the DNA sequences given below,oligonucleotide primers can be synthesized that are complementary to theknown polynucleotide sequence. These primers can then be used in PCR toamplify the polynucleotide from template derived from mRNA or genomicDNA isolated from plant material. The resulting amplified products canthen be cloned into commercially available cloning vectors, such as theTA series of vectors from InVitrogen. By sequencing the individualclones thus identified with sequencing primers designed from theoriginal sequence, it is then possible to extend the sequence in bothdirections to determine the full gene sequence. Such sequencing isperformed using denatured double stranded DNA prepared from a plasmidclone. Suitable techniques are described by Maniatis, T., Fritsch, E. F.and Sambrook, J. in MOLECULAR CLONING, A Laboratory Manual (2nd edition1989 Cold Spring Harbor Laboratory. See Sequencing DenaturedDouble-Stranded DNA Templates 13.70). Illustrative of the invention, thepolynucleotide set out below were assembled from a cDNA library derivedfor example, from germinating maize seeds.

[0081] Myo-inositol 1-phosphate synthase of the present invention isstructurally related to other proteins of the myo-inositol 1-phosphatesynthase family, as shown by comparing the present sequence encodingmyo-inositol 1-phosphate synthase with sequences reported in theliterature. A preferred DNA sequence is set out below. It contains anopen reading frame encoding a protein of about 510 amino acid residueswith a deduced molecular weight of about 59.7 (calculated as the numberof amino acid residues X 117) kDa. The protein exhibits greatesthomology to myo-inositol-1-phosphate synthase. The present myo-inositol1-phosphate synthase has about 88% identity and about 92% similaritywith the amino acid sequence of myo-inositol-1-phosphate synthase fromMesembryantherum crystallium and 78.7% identity at the nucleic acidlevel. (These percentages are based on comparison of full-length codingsequence only, i.e., ATG through stop codon).

[0082] Myo-inositol monophosphatase-3of the invention is structurallyrelated to other proteins of the myo-inositol monophosphatase-3family,as shown by comparing the present sequence encoding myo-inositolmonophosphatase-3with that of sequence reported in the literature. Apreferred DNA sequence is set out below. It contains an open readingframe encoding a protein of about 267 amino acid residues with a deducedmolecular weight of about 31.2 kDa (calculated as the number of aminoacid residues X 117). Novel myo-inositol monophosphatase-3 identified byhomology between the amino acid sequence set out below and known aminoacid sequences of other proteins such as myo-inositolmonophosphatase-3from Lycopersicum esulentum with 76.1% identity/81.1%similarity at the amino acid level and 67.9% identity at the nucleicacid level. (These percentages are based on comparison of full-lengthcoding sequence only i.e., ATG through stop codon).

[0083] Myo-inositol 1,3,4-trisphosphate 5/6-kinase of the invention isstructurally related to other proteins of the myo-inositol1,3,4-trisphosphate 5/6-kinase family, as shown by comparing thesequence encoding the present inositol 1,3,4-trisphosphate 5/6-kinasewith that of sequence reported in the literature. A preferred DNAsequence is set out below. It contains an open reading frame encoding aprotein of about 353 amino acid residues with a deduced molecular weightof about 41.3 kDa (calculated as the number of amino acid residues X117). The protein exhibits greatest homology to myo-inositol1,3,4-trisphosphate 5/6-kinase from Homo sapiens. myo-inositol1,3,4-trisphosphate 5/6-kinase below has about 34% identity and about43.4% similarity with the amino acid sequence of myo-inositol1,3,4-trisphosphate 5/6-kinase from Homo sapiens. (The percentagesdisclosed above are based on comparison of full-length coding sequenceonly i.e., ATG through stop codon.)

[0084] A preferred phosphatidylinositol 3-kinase sequence is set outbelow. It contains an open reading frame encoding a protein of about 803amino acid residues with a deduced molecular weight of about 94.1 kDa(calculated as the number of amino acid residues X 117). The proteinexhibits greatest homology to phosphatidylinositol 3-kinase from Glycinemax. Homology between amino acid sequences set out in the followingsequences and known amino acid sequences of other proteins such asphosphatidylinositol 3-kinase from Glycine max with 78% identity/84%similarity at the amino acid level and 73% identity at the nucleic acidlevel (these percentages are based on comparison of full-length codingsequence only i.e., ATG through stop codon) based on the Gap programdefined below.

[0085] Polynucleotides of the present invention may be in the form ofRNA, such as mRNA, or in the form of DNA, including, for instance, cDNAand genomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded DNA may be the coding strand, alsoknown as the sense strand, or it may be the non-coding strand, alsoreferred to as the antisense strand.

[0086] The coding sequence which encodes the polypeptide may beidentical to the coding sequence of the polynucleotides shown below. Italso may be a polynucleotide with a different sequence, which, as aresult of the redundancy (degeneracy) of the genetic code, encodes thepolypeptides shown below. As discussed more fully below, thesealternative coding sequences are an important source of sequences forcodon optimization.

[0087] Polynucleotides of the present invention which encode thepolypeptides listed below may include, but are not limited to the codingsequence for the mature polypeptide, by itself; the coding sequence forthe mature polypeptide and additional coding sequences, such as thoseencoding a leader or secretory sequence, such as a pre-, or pro- orprepro-protein sequence; the coding sequence of the mature polypeptide,with or without the aforementioned additional coding sequences, togetherwith additional, non-coding sequences, including for example, but notlimited to non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription (includingtermination signals, for example), ribosome binding, mRNA stabilityelements, and additional coding sequence which encode additional aminoacids, such as those which provide additional functionalities.

[0088] The DNA may also comprise promoter regions which function todirect the transcription of the mRNA encoding phytate biosyntheticenzymes of this invention. Such promoters may be independently useful todirect the transcription of heterologous genes in recombinant expressionsystems. Heterologous is defined as a sequence that is not naturallyoccurring with the promoter sequence. While the nucleotide sequence isheterologous to the promoter sequence, it may be homologous, or native,or heterologous, or foreign to the plant host.

[0089] Furthermore, the polypeptide may be fused to a marker sequence,such as a peptide, which facilitates purification of the fusedpolypeptide. In certain embodiments of this aspect of the invention, themarker sequence is a hexa-histidine peptide, such as the tag provided inthe pQE vector (Qiagen, Inc.) and the pET series of vectors (Novagen),among others, many of which are commercially available. As described inGentz et al., Proc. Nat'l. Acad. Sci., (USA) 86:821-824 (1989), forinstance, hexa-histidine provides for convenient purification of thefusion protein. The HA tag may also be used to create fusion proteinsand corresponds to an epitope derived of influenza hemagglutin inprotein, which has been described by Wilson et al., Cell 37:767 (1984),for instance.

[0090] In accordance with the foregoing, the term “polynucleotideencoding a polypeptide” as used herein encompasses polynucleotides whichinclude a sequence encoding a polypeptide of the present invention,particularly plant, and more particularly Zea mays phytate biosyntheticenzymes having the amino acid sequence set out below. The termencompasses polynucleotides that include a single continuous region ordiscontinuous regions encoding the polypeptide (for example, interruptedby integrated phage or insertion sequence or editing) together withadditional regions, that also may contain coding and/or non-codingsequences.

[0091] The present invention further relates to variants of the presentpolynucleotides which encode for fragments, analogs and derivatives ofthe polypeptides having the deduced amino acid sequence below. A variantof the polynucleotide may be a naturally occurring variant such as anaturally occurring allelic variant, or it may be a variant that is notknown to occur naturally. Such non-naturally occurring variants of thepolynucleotide may be made by mutagenesis techniques, including thoseapplied to polynucleotides, cells or organisms.

[0092] Among variants in this regard are variants that differ from theaforementioned polynucleotides by nucleotide substitutions, deletions oradditions. The substitutions may involve one or more nucleotides. Thevariants may be altered in coding or non-coding regions or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.

[0093] Among the particularly preferred embodiments of the invention inthis regard are polynucleotides encoding polypeptides having the aminoacid sequences set out below; variants, analogs, derivatives andfragments thereof.

[0094] Further particularly preferred in this regard are polynucleotidesencoding phytate biosynthetic enzyme variants, analogs, derivatives andfragments, and variants, analogs and derivatives of the fragments, whichhave the amino acid sequences below in which several, a few, 1 to 10, 1to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted oradded, in any combination. Especially preferred among these are silentsubstitutions, additions and deletions, which do not alter theproperties and activities of the phytate biosynthetic enzymes. Alsoespecially preferred in this regard are conservative substitutions. Mosthighly preferred are polynucleotides encoding polypeptides having theamino acid sequence below, without substitutions.

[0095] Further preferred embodiments of the invention arepolynucleotides that are greater than 79%, preferably at least 80%, morepreferably at least 85% identical to a polynucleotide encodingmyo-inositol 1-phosphate synthase polypeptide having the amino acidsequence set out below, and polynucleotides which are complementary tosuch polynucleotides. Among these particularly preferredpolynucleotides, those with at least 90%, 95%, 98% or at least 99% areespecially preferred.

[0096] Further preferred embodiments of the invention arepolynucleotides that are greater than 70%, preferably at least 75%, morepreferably at least 80% identical to a polynucleotide encodingmyo-inositol monophosphatase-3 polypeptide having the amino acidsequence set out below, and polynucleotides which are complementary tosuch polynucleotides. Among these particularly preferredpolynucleotides, those with at least 85%, 90%, 95%, 98% or at least 99%are especially preferred.

[0097] Further preferred embodiments of the invention arepolynucleotides that are greater than 45%, preferably at least 50%, morepreferably at least 55%, still more preferably at least 60% identical toa polynucleotide encoding myo-inositol 1,3,4-triphosphate 5/6-kinasepolypeptide having the amino acid sequence set out below, andpolynucleotides which are complementary to such polynucleotides. Amongthese particularly preferred polynucleotides, those with at least 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% are especiallypreferred.

[0098] Further preferred embodiments of the invention arepolynucleotides that are greater than 73%, preferably at least 75%, morepreferably at least 80% identical to a polynucleotide encodingphosphatidylinositol 3-kinase polypeptide having the amino acid sequenceset out below, and polynucleotides which are complementary to suchpolynucleotides. Among these particularly preferred polynucleotides,those with at least 85%, 90%, 95%, 98% or at least 99% are especiallypreferred.

[0099] Particularly preferred embodiments in this respect, moreover, arepolynucleotides which encode polypeptides which retain substantially thesame or even exhibit a reduction in the biological function or activityas the mature polypeptide encoded by the polynucleotides set out below.

[0100] The present invention further relates to polynucleotides thathybridize to the herein above-described sequences. In this regard, thepresent invention especially relates to polynucleotides which hybridizeunder stringent conditions to the herein above-describedpolynucleotides. As herein used, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

[0101] The terms “stringent conditions” or “stringent hybridizationconditions” includes reference to conditions under which a probe willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

[0102] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, I M NaCl, 1% SDS (sodium dodecyl sulfate) at 37° C.,and a wash in 1× to 2× SSC (20× SSC=3.0 M NaCl/0.3 M trisodium citrate)at 50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.5× to 1× SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1× SSC at 60 to 65° C.

[0103] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138:267-284 (1984): T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, %GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridizationand/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

[0104] As discussed additionally herein regarding polynucleotide assaysof the invention, for instance, polynucleotides of the invention asdiscussed above, may be used as a hybridization probe for RNA, cDNA andgenomic DNA to isolate full-length cDNAs and genomic clones encodingphytate biosynthetic enzymes and to isolate cDNA and genomic clones ofother genes that have a high sequence similarity to the genes. Suchprobes generally will comprise at least 15 bases. Preferably, suchprobes will have at least 30 bases and may have at least 50 bases.Particularly preferred probes will have at least 30 bases and will have50 bases or less.

[0105] The polynucleotides and polypeptides of the present invention maybe employed as research reagents and materials for discovery of highphosphorous transgenic corn plants. The polynucleotides of the inventionthat are oligonucleotides, derived from the sequences below may be usedas PCR primers in the process herein described to determine whether ornot the genes identified herein in whole or in part are transcribed inphytic acid accumulating tissue.

[0106] The polynucleotides may encode a polypeptide which is the matureprotein plus additional amino or carboxyl-terminal amino acids, or aminoacids interior to the mature polypeptide (when the mature form has morethan one polypeptide chain, for instance). Such sequences may play arole in processing of a protein from precursor to a mature form, mayallow protein transport, may lengthen or shorten protein half-life ormay facilitate manipulation of a protein for assay or production, amongother things. As generally is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.

[0107] A precursor protein, having the mature form of the polypeptidefused to one or more prosequences may be an inactive form of thepolypeptide. When prosequences are removed such inactive precursorsgenerally are activated. Some or all of the prosequences may be removedbefore activation. Generally, such precursors are called proproteins.

[0108] In sum, a polynucleotide of the present invention may encode amature protein, a mature protein plus a leader sequence (which may bereferred to as a preprotein), a precursor of a mature protein having oneor more prosequences which are not the leader sequences of a preprotein,or a preproprotein, which is a precursor to a proprotein, having aleader sequence and one or more prosequences, which generally areremoved during processing steps that produce active and mature forms ofthe polypeptide.

[0109] Polypeptides

[0110] The present invention further relates to polypeptides that havethe deduced amino acid sequences below.

[0111] The invention also relates to fragments, analogs and derivativesof these polypeptides. The terms “fragment,” “derivative” and “analog”when referring to the polypeptides, means a polypeptide which retainsessentially the same biological function or activity as suchpolypeptide. Fragments derivatives and analogs that retain at least 90%of the activity of the native phytate biosynthetic enzymes arepreferred. Fragments, derivatives and analogs that retain at least 95%of the activity of the native polypeptides are preferred. Thus, ananalog includes a proprotein which can be activated by cleavage of theproprotein portion to produce an active mature polypeptide.

[0112] The polypeptide of the present invention may be a recombinantpolypeptide, a natural polypeptide or a synthetic polypeptide. Incertain preferred embodiments it is a recombinant polypeptide.

[0113] The fragment, derivative or analog of the polypeptides below maybe (i) one in which one or more of the amino acid residues aresubstituted with a conserved or non-conserved amino acid residue(preferably a conserved amino acid residue) and such substituted aminoacid residue may or may not be one encoded by the genetic code, or (ii)one in which one or more of the amino acid residues includes asubstituent group, or (iii) one in which the mature polypeptide is fusedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol), or (iv) one in whichthe additional amino acids are fused to the mature polypeptide, such asa leader or secretory sequence or a sequence which is employed forpurification of the mature polypeptide or a proprotein sequence. Suchfragments, derivatives and analogs are deemed to be obtained by those ofordinary skill in the art, from the teachings herein.

[0114] Among the particularly preferred embodiments of the invention inthis regard are polypeptides having the amino acid sequence of phytatebiosynthetic enzymes set out below, variants, analogs, derivatives andfragments thereof, and variants, analogs and derivatives of thefragments.

[0115] Among preferred variants are those that vary from a reference byconservative amino acid substitutions. Such substitutions are those thatsubstitute a given amino acid in a polypeptide by another amino acid oflike characteristics. Typically seen as conservative substitutions arethe replacements, one for another, among the aliphatic amino acids Ala,Val, Leu and lie; interchange of the hydroxyl residues Ser and Thr,exchange of the acidic residues Asp and Glu, substitution between theamide residues Asn and Gln, exchange of the basic residues Lys and Argand replacements among the aromatic residues Phe, Tyr.

[0116] Further particularly preferred in this regard are variants,analogs, derivatives and fragments, and variants, analogs andderivatives of the fragments, having the amino acid sequence below, inwhich several, a few, 1 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acidresidues are substituted, deleted or added, in any combination.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of thephytate biosynthetic enzymes. Also especially preferred in this regardare conservative substitutions. Most highly preferred are polypeptideshaving the amino acid sequences below without substitutions.

[0117] The polypeptides and polynucleotides of the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

[0118] The polypeptides of the present invention include themyo-inositol 1-phosphate synthase polypeptide (in particular the maturepolypeptide) as well as polypeptides which have greater than 88%identity (92% similarity) to the polypeptide, as described above inNeedleman and Wunsch, and more preferably at least 90% identity (95%similarity), still more preferably at least 95% identity (98%similarity) and most preferably at least 98% identity and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0119] The polypeptides of the present invention include themyo-inositol monophosphatase-3 polypeptide (in particular the maturepolypeptide) as well as polypeptides which have greater than 77%identity (82% similarity) to the polypeptide, as described above inNeedleman and Wunsch, more preferably at least 80% identity (85%similarity), still more preferably at least 85% identity (90%similarity), still more preferably at least 90% identity (95%similarity), still more preferably at least 95% identity (98%similarity) and most preferably at least 98% identity and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0120] The polypeptides of the present invention include themyo-inositol 1,3,4-triphosphate 5/6-kinase polypeptide (in particularthe mature polypeptide) as well as polypeptides which have greater than35% identity (45% similarity) to the polypeptide, as described above inNeedleman and Wunsch, more preferably at least 50% identity (60%similarity), still more preferably at least 60% identity (70%similarity), more preferably at least 80% identity (85% similarity),still more preferably at least 70% identity (80% similarity), morepreferably at least 80% identity (85% similarity), still more preferablyat least 85% identity (90% similarity), still more preferably at least90% identity (95% similarity), still more preferably at least 95%identity (98% similarity) and most preferably at least 98% identity andalso include portions of such polypeptides with such portion of thepolypeptide generally containing at least 30 amino acids and morepreferably at least 50 amino acids.

[0121] The polypeptides of the present invention include thephosphatidylinositol 3-kinase polypeptide (in particular the maturepolypeptide) as well as polypeptides which have greater than 78%identity (84% similarity) to the polypeptide, as described above inNeedleman and Wunsch, more preferably at least 80% identity (85%similarity), still more preferably at least 85% identity (90%similarity), still more preferably at least 90% identity (95%similarity), still more preferably at least 95% identity (98%similarity) and most preferably at least 98% identity and also includeportions of such polypeptides with such portion of the polypeptidegenerally containing at least 30 amino acids and more preferably atleast 50 amino acids.

[0122] Vectors, Host Cells, Expression

[0123] The present invention also relates to vectors comprising thepolynucleotides of the present invention, host cells that incorporatethe vectors of the invention and the production of polypeptides of theinvention by recombinant techniques.

[0124] Host cells can be genetically engineered to incorporate thepolynucleotides and express polypeptides of the present invention. Forinstance, the polynucleotides may be introduced into host cells usingwell known techniques of infection, transduction, transfection,transvection and transformation. The polynucleotides may be introducedalone or with other polynucleotides. Such other polynucleotides may beintroduced independently, co-introduced or introduced joined to thepolynucleotides of the invention.

[0125] Thus, for instance, polynucleotides of the invention may betransfected into host cells with another, separate, polynucleotideencoding a selectable marker, using standard techniques forco-transfection and selection in, for instance, plant cells. In thiscase the polynucleotides generally will be stably incorporated into thehost cell genome.

[0126] Alternatively, the polynucleotides may be joined to a vectorcontaining a selectable marker for propagation in a host. The vectorconstruct may also be introduced into host cells by the aforementionedtechniques. Generally, a plasmid vector is introduced as DNA in aprecipitate, such as a calcium phosphate precipitate, or in a complexwith a charged lipid. Electroporation also may be used to introducepolynucleotides into a host. If the vector is a virus, it may bepackaged in vitro or introduced into a packaging cell and the packagedvirus may be transduced into cells. A wide variety of techniquessuitable for making polynucleotides and for introducing polynucleotidesinto cells in accordance with this aspect of the invention are wellknown and routine to those of skill in the art. Such techniques arereviewed at length in Sambrook et al., cited above, which isillustrative of the many laboratory manuals that detail thesetechniques.

[0127] Vectors

[0128] In accordance with this aspect of the invention the vector maybe, for example, a plasmid vector, a single or double-stranded phagevector, a single or double-stranded RNA or DNA viral vector. Suchvectors may be introduced into cells as polynucleotides, preferably DNA,by well known techniques for introducing DNA and RNA into cells. Thevectors, in the case of phage and viral vectors also may be andpreferably are introduced into cells as packaged or encapsidated virusby well known techniques for infection and transduction. Viral vectorsmay be replication competent or replication defective. In the lattercase viral propagation generally will occur only in complementing hostcells.

[0129] Preferred among vectors, in certain respects, are those forexpression of polynucleotides and polypeptides of the present invention.Generally, such vectors comprise cis-acting control regions effectivefor expression in a host operatively linked to the polynucleotide to beexpressed. Appropriate trans-acting factors either are supplied by thehost, supplied by a complementing vector or supplied by the vectoritself upon introduction into the host.

[0130] In certain preferred embodiments in this regard, the vectorsprovide for preferred expression. Such preferred expression may beinducible expression or expression predominantly in certain types ofcells or both inducible and cell-preferred. Particularly preferred amonginducible vectors are vectors that can be induced for expression byenvironmental factors that are easy to manipulate, such as temperatureand nutrient additives. A variety of vectors suitable to this aspect ofthe invention, including constitutive and inducible expression vectorsfor use in prokaryotic and eukaryotic hosts, are well known and employedroutinely by those of skill in the art. Such vectors include, amongothers, chromosomal, episomal and virus-derived vectors, e.g., vectorsderived from bacterial plasmids, from bacteriophage, from transposons,from yeast episomes, from insertion elements, from yeast chromosomalelements, from viruses such as baculoviruses, papova viruses, such asSV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabiesviruses and retroviruses, and vectors derived from combinations thereof,such as those derived from plasmid and bacteriophage genetic elements,such as cosmids and phagemids and binaries used forAgrobacterium-mediated transformations. All may be used for expressionin accordance with this aspect of the present invention. Generally, anyvector suitable to maintain, propagate or express polynucleotides toexpress a polypeptide in a host may be used for expression in thisregard.

[0131] The following vectors, which are commercially available, areprovided by way of example. Among vectors preferred for use in bacteriaare pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors,Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A,available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,pRIT5 available from Pharmacia. Among preferred eukaryotic vectors arepWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; andpSVK3, pBPV, pMSG and pSVL available from Pharmacia. Useful plantbinaries vectors include BIN19 and its derivatives available fromClontech. These vectors are listed solely by way of illustration of themany commercially available and well known vectors that are available tothose of skill in the art for use in accordance with this aspect of thepresent invention. It will be appreciated that any other plasmid orvector suitable for, for example, introduction, maintenance, propagationor expression of a polynucleotide or polypeptide of the invention in ahost may be used in this aspect of the invention.

[0132] In general, expression constructs will contain sites fortranscription initiation and termination, and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will include atranslation initiating AUG at the beginning and a termination codonappropriately positioned at the end of the polypeptide to be translated.

[0133] In addition, the constructs may contain control regions thatregulate as well as engender expression. Generally, in accordance withmany commonly practiced procedures, such regions will operate bycontrolling transcription, such as transcription factors, repressorbinding sites and termination, among others. For secretion of thetranslated protein into the lumen of the endoplasmic reticulum, into theperiplasmic space or into the extracellular environment, appropriatesecretion signals may be incorporated into the expressed polypeptide.These signals may be endogenous to the polypeptide or they may beheterologous signals.

[0134] Generally, recombinant expression vectors will include origins ofreplication, a promoter derived from a highly-expressed gene to directtranscription of a downstream structural sequence, and a selectablemarker to permit isolation of vector containing cells after exposure tothe vector.

[0135] Transcription of the DNA encoding the polypeptides of the presentinvention by higher eukaryotes may be increased by inserting an enhancersequence into the vector. Enhancers are cis-acting elements of DNA,usually about from 10 to 300 bp that act to increase transcriptionalactivity of a promoter in a given host cell-type. Examples of enhancersinclude the SV40 enhancer, which is located on the late side of thereplication origin at bp 100 to 270, the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. Additional enhancers useful in theinvention to increase transcription of the introduced DNA segment,include, inter alia, viral enhancers like those within the 35S promoter,as shown by Odell et al., Plant Mol. Biol. 10:263-72 (1988), and anenhancer from an opine gene as described by Fromm et al., Plant Cell1:977 (1989).

[0136] Among known eukaryotic promoters suitable in this regard are theCMV immediate early promoter, the HSV thymidine kinase promoter, theearly and late SV40 promoters, the promoters of retroviral LTRs, such asthose of the Rous sarcoma virus (“RSV”), metallothionein promoters, suchas the mouse metallothionein-I promoter and various plant promoters,such as globulin-1. When available, the native promoters of the phytatebiosynthetic enzyme genes may be used.

[0137] As mentioned above, the DNA sequence in the expression vector isoperatively linked to appropriate expression control sequence(s),including, for instance, a promoter to direct mRNA transcription.Representatives of prokaryotic promoters include the phage lambda PLpromoter, the E. coli lac, trp and tac promoters to name just a few ofthe well-known promoters.

[0138] With respect to plants, examples of seed-specific promotersinclude promoters of seed storage proteins which express these proteinsin seeds in a highly regulated manner (Thompson et al.; BioEssays.10:108; (1989), incorporated herein in its entirety by reference), suchas, for dicotyledonous plants, a bean β-phaseolin promoter, a napinpromoter, a β-conglycinin promoter, and a soybean lectin promoter. Formonocotyledonous plants, promoters useful in the practice of theinvention include, but are not limited to, a maize 15 kD zein promoter,a 22 kD zein promoter, a γ-zein promoter, a waxy promoter, a shrunken 1promoter, a globulin 1 promoter, and the shrunken 2 promoter. However,other promoters useful in the practice of the invention are known tothose of skill in the art.

[0139] Other examples of suitable promoters are the promoter for thesmall subunit of ribulose-1,5-bis-phosphatecarboxylase, promoters fromtumor-inducing plasmids of Agrobacterium tumefaciens, such as thenopaline synthase and octopine synthase promoters, and viral promoterssuch as the cauliflower mosaic virus (CaMV) 19S and 35S promoters or thefigwort mosaic virus 35S promoter.

[0140] It will be understood that numerous promoters not mentioned aresuitable for use in this aspect of the invention are well known andreadily may be employed by those of skill in the manner illustrated bythe discussion and the examples herein. For example this inventioncontemplates using the native phytate biosynthetic enzyme promoters todrive the expression of the enzyme in a recombinant environment.

[0141] Vectors for propagation and expression generally will includeselectable markers. Such markers also may be suitable for amplificationor the vectors may contain additional markers for this purpose. In thisregard, the expression vectors preferably contain one or more selectablemarker genes to provide a phenotypic trait for selection of transformedhost cells. Preferred markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture, and tetracycline orampicillin resistance genes for culturing E. coli and other prokaryotes.Kanamycin and herbicide resistance genes (PAT and BAR) are generallyuseful in plant systems.

[0142] Selectable marker genes, in physical proximity to the introducedDNA segment, are used to allow transformed cells to be recovered byeither positive genetic selection or screening. The selectable markergenes also allow for maintaining selection pressure on a transgenicplant population, to ensure that the introduced DNA segment, and itscontrolling promoters and enhancers, are retained by the transgenicplant.

[0143] Many of the commonly used positive selectable marker genes forplant transformation have been isolated from bacteria and code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide. Other positive selection marker genesencode an altered target which is insensitive to the inhibitor.

[0144] A preferred selection marker gene for plant transformation is theBAR or PAT gene, which is used with the selecting agent bialaphos.Spencer et al., T. Thero. Appl'd Genetics 79:625-631 (1990). Anotheruseful selection marker gene is the neomycin phosphotransferase II(nptII) gene, isolated from Tn5, which confers resistance to kanamycinwhen placed under the control of plant regulatory signals. Fraley etal., Proc. Nat'l Acad. Sci. (USA) 80:4803 (1983). The hygromycinphosphotransferase gene, which confers resistance to the antibiotichygromycin, is a further example of a useful selectable marker. VandenElzen et al., Plant Mol. Biol. 5:299 (1985). Additional positiveselectable markers genes of bacterial origin that confer resistance toantibiotics include gentamicin acetyl transferase, streptomycinphosphotransferase, aminoglycoside-3′-adenyltransferase and thebleomycin resistance determinant. Hayford et al., Plant Physiol. 86:1216(1988); Jones et al., Mol. Gen. Genet. 210:86 (1987); Svab et al., PlantMol. Biol. 14:197 (1990); Hille et al., Plant Mol. Biol. 7:171 (1986).

[0145] Other positive selectable marker genes for plant transformationare not of bacterial origin. These genes include mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphatesynthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987); Shah et al., Science 233:478 (1986); Charest et al., Plant CellRep. 8:643 (1990).

[0146] Another class of useful marker genes for plant transformationwith the DNA sequence requires screening of presumptively transformedplant cells rather than direct genetic selection of transformed cellsfor resistance to a toxic substance such as an antibiotic. These genesare particularly useful to quantitate or visualize the spatial patternof expression of the DNA sequence in specific tissues and are frequentlyreferred to as reporter genes because they can be fused to a gene orgene regulatory sequence for the investigation of gene expression.Commonly used genes for screening presumptively transformed cellsinclude β-glucuronidase (GUS), β-galactosidase, luciferase, andchloramphenicol acetyltransferase. Jefferson, Plant Mol. Biol. Rep.5:387 (1987); Teeri et al., EMBO J. 8:343 (1989); Koncz et al., Proc.Nat'l Acad. Sci. (USA) 84:131 (1987); De Block et al., EMBO J. 3:1681(1984). Another approach to the identification of relatively raretransformation events has been use of a gene that encodes a dominantconstitutive regulator of the Zea mays anthocyanin pigmentation pathway(Ludwig et al., Science 247:449 (1990)).

[0147] The appropriate DNA sequence may be inserted into the vector byany of a variety of well-known and routine techniques. In general, a DNAsequence for expression is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionendonucleases and then joining the restriction fragments together usingT4 DNA ligase. The sequence may be inserted in a forward or reverseorientation. Procedures for restriction and ligation that can be used tothis end are well known and routine to those of skill. Suitableprocedures in this regard, and for constructing expression vectors usingalternative techniques, which also are well known and routine to thoseskill, are set forth in great detail in Sambrook et al., MOLECULARCLONING, A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1989).

[0148] Polynucleotides of the invention, encoding the heterologousstructural sequence of a polypeptide of the invention generally will beinserted into the vector using standard techniques so that it isoperably linked to the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG that initiates translation of the polypeptide tobe expressed. Generally, there will be no other open reading frames thatbegin with an initiation codon, usually AUG, and lie between theribosome binding site and the initiation codon. Also, generally, therewill be a translation stop codon at the end of the polypeptide and therewill be a polyadenylation signal in constructs for use in eukaryotichosts. Transcription termination signal appropriately disposed at the 3′end of the transcribed region may also be included in the polynucleotideconstruct.

[0149] The vector containing the appropriate DNA sequence as describedelsewhere herein, as well as an appropriate promoter, and otherappropriate control sequences, may be introduced into an appropriatehost using a variety of well known techniques suitable to expressiontherein of a desired polypeptide. The present invention also relates tohost cells containing the above-described constructs discussed. The hostcell can be a higher eukaryotic cell, such as a mammalian or plant cell,or a lower eukaryotic cell, such as a yeast cell, or the host cell canbe a prokaryotic cell, such as a bacterial cell.

[0150] Introduction of the construct into the host cell can be effectedby calcium phosphate transfection, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) andSambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

[0151] Representative examples of appropriate hosts include bacterialcells, such as streptococci, staphylococci, E. coli, streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells andAspergillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9cells; animal cells such as CHO, COS and Bowes melanoma cells; and plantcells. Hosts for a great variety of expression constructs are wellknown, and those of skill will be enabled by the present disclosurereadily to select a host for expressing a polypeptide in accordance withthis aspect of the present invention.

[0152] The engineered host cells can be cultured in conventionalnutrient media, which may be modified as appropriate for, inter alia,activating promoters, selecting transformants or amplifying genes.Culture conditions, such as temperature, pH and the like, previouslyused with the host cell selected for expression generally will besuitable for expression of polypeptides of the present invention as willbe apparent to those of skill in the art.

[0153] Constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence.Alternatively, the polypeptides of the invention can be syntheticallyproduced by conventional peptide synthesizers.

[0154] Mature proteins can be expressed in mammalian cells, yeast,bacteria, or other cells under the control of appropriate promoters.Cell-free translation systems can also be employed to produce suchproteins using RNAs derived from the DNA constructs of the presentinvention.

[0155] Following transformation of a suitable host strain and growth ofthe host strain to an appropriate cell density, where the selectedpromoter is inducible it is induced by appropriate means (e.g.,temperature shift or exposure to chemical inducer) and cells arecultured for an additional period.

[0156] Cells typically then are harvested by centrifugation, disruptedby physical or chemical means, and the resulting crude extract retainedfor further purification. Microbial cells employed in expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents, such methods are well know to those skilled in the art.

[0157] As noted above, the present invention provides vectors capable ofexpressing phytate biosynthetic enzymes under the control of suitablepromoters. In general, the vectors should be functional in plant cells.At times, it may be preferable to have vectors that are functional in E.coli (e.g., production of protein for raising antibodies, DNA sequenceanalysis, construction of inserts, obtaining quantities of nucleic acidsand proteins). Vectors and procedures for cloning and expression in E.coli are discussed above and, for example, in Sambrook et al. (supra)and in Ausubel et al. (supra).

[0158] Vectors that are functional in plants are preferably binaryplasmids derived from Agrobacterium plasmids. Such vectors are capableof transforming plant cells. These vectors contain left and right bordersequences that are required for integration into the host (plant)chromosome. At minimum, between these border sequences is the gene to beexpressed under control of a promoter. In preferred embodiments, aselectable marker and a reporter gene are also included. For ease ofobtaining sufficient quantities of vector, a bacterial origin thatallows replication in E. coli is preferred.

[0159] In certain preferred embodiments, the vector contains a reportergene and the structural genes of this invention. The reporter geneshould allow ready determination of transformation and expression. TheGUS (β-glucuronidase) gene is preferred (U.S. Pat. No. 5,268,463). Otherreporter genes, such as β-galactosidase, luciferase, GFP, and the like,are also suitable in the context of this invention. Methods andsubstrates for assaying expression of each of these genes are well knownin the art. The reporter gene should be under control of a promoter thatis functional in plants. Such promoters include CaMV 35S promoter,mannopine synthase promoter, ubiquitin promoter and DNA J promoter.

[0160] Preferably, the vector contains a selectable marker foridentifying transformants. The selectable marker may confer a growthadvantage under appropriate conditions. Generally, selectable markersare drug resistance genes, such as neomycin phosphotransferase. Otherdrug resistance genes are known to those in the art and may be readilysubstituted. The selectable marker has a linked constitutive orinducible promoter and a termination sequence, including apolyadenylation signal sequence.

[0161] Additionally, a bacterial origin of replication and a selectablemarker for bacteria are preferably included in the vector. Of thevarious origins (e.g., coIEI, fd phage), a coIEI origin of replicationis preferred. Most preferred is the origin from the pUC plasmids, whichallow high copy number.

[0162] A general vector suitable for use in the present invention isbased on pBI121 (U.S. Pat. No. 5,432,081) a derivative of pBIN19. Othervectors have been described (U.S. Pat. No. 4,536,475) or may beconstructed based on the guidelines presented herein. The plasmid pBI121contains a left and right border sequence for integration into a planthost chromosome. These border sequences flank two genes. One is akanamycin resistance gene (neomycin phosphotransferase) driven by anopaline synthase promoter and using a nopaline synthase polyadenylationsite. The second is the E. coli GUS gene under control of the CaMV 35Spromoter and polyadenylated using a nopaline synthase polyadenylationsite. Plasmid pBI121 also contains a bacterial origin of replication andselectable marker.

[0163] In certain embodiments, the vector may contain the structuralgenes identified herein under control of a promoter. The promoter may bethe native promoters associated with the structural genes themselves ora strong, constitutive promoter, such as CaMV 35S promoter. Otherelements that are preferred for optimal expression (e.g., transcriptiontermination site, enhancer, splice site) may also be included. The genesmay alternatively be expressed as fusion proteins with a reporter gene,for example.

[0164] Plant Transformation Methods

[0165] As discussed above the present invention also provides methodsfor producing a plant which expresses a foreign gene, comprising thesteps of (a) introducing a vector as described above into an embryogenicplant cell, wherein the vector contains a foreign gene in an expressibleform, and (b) producing a plant from the embryogenic plant cell, whereinthe plant expresses the foreign gene.

[0166] Vectors may be introduced into plant cells by any of severalmethods. For example, DNA may be introduced as a plasmid byAgrobacterium in co-cultivation or bombardment. Other transformationmethods include electroporation, CaPO₄-mediated transfection, and thelike. Preferably, DNA is first transfected into Agrobacterium andsubsequently introduced into plant cells. Most preferably, the infectionis achieved by co-cultivation. In part, the choice of transformationmethods depends upon the plant to be transformed.

[0167] Phytate biosynthetic polypeptides can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Well known techniques for refolding protein may be employed toregenerate active conformation when the polypeptide is denatured duringisolation and or purification.

[0168] Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

[0169] It is appreciated that the gene expressing the polypeptide ofinterest may have to be “codon-optimized” to affect efficient expressionof a particular host. Thus, this invention contemplates selecting fromthe sequences below, the particular codon optimized sequence for theparticular host cell of interest.

[0170] Other genes of interest may be “stacked” during the sametransformation events. For example, other genes of interest may impartdisease, pest or herbicide resistance, or improve the feed and foodquality of the plant or seed, such increased or altered oil expressionor altered protein or carbohydrate expression.

[0171] Regeneration of Transformed Plants

[0172] Following transformation, regeneration is involved to obtain awhole plant from transformed cells. Techniques for regenerating plantsfrom tissue culture such as transformed protoplasts or callus celllines, are known in the art. For example, see Phillips et al.; PlantCell Tissue Organ Culture; Vol. 1: p 123; (1981); Patterson et al.;Plant Sci.; Vol. 42; p. 125; (1985); Wright et al.; Plant Cell Reports;Vol. 6: p. 83; (1987); and Barwale et al.; Planta; Vol. 167; p. 473(1986); each incorporated herein in its entirety by reference. Theselection of an appropriate method is within the skill of the art.

[0173] It is expected that the transformed plants will be used intraditional breeding programs, including TOPCROSS pollination systems asdisclosed in U.S. Pat. No. 5,706,603 and U.S. Pat. No. 5,704,160 thedisclosure of each is incorporated herein by reference.

[0174] Polynucleotide Assays

[0175] This invention is also related to the use of the phytatebiosyntheticenzyme polynucleotides in marker to assist in breedingprogram, as described for example in PCT publication US89/00709. The DNAmay be used directly for detection or may be amplified enzymatically byusing PCR prior to analysis. PCR (Saiki et al., Nature 324:163-166(1986)). RNA or cDNA may also be used in the same ways. As an example,PCR primers complementary to the nucleic acid encoding the phytatebiosynthetic enzymes can be used to identify and analyze phytatebiosynthetic enzyme presence and expression. Using PCR, characterizationof the gene present in a particular tissue or plant variety may be madeby an analysis of the genotype of the tissue or variety. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the genotype of a reference sequence.Point mutations can be identified by hybridizing amplified DNA toradiolabeled phytate biosynthetic enzyme RNA or alternatively,radiolabeled phytate biosynthetic enzyme antisense DNA sequences.Perfectly matched sequences can be distinguished from mismatchedduplexes by RNase A digestion or by differences in melting temperatures.

[0176] Sequence differences between a reference gene and genes havingmutations also may be revealed by direct DNA sequencing. In addition,cloned DNA segments may be employed as probes to detect specific DNAsegments. The sensitivity of such methods can be greatly enhanced byappropriate use of PCR or another amplification method. For example, asequencing primer is used with double-stranded PCR product or asingle-stranded template molecule generated by a modified PCR. Thesequence determination is performed by conventional procedures withradiolabeled nucleotide or by automatic sequencing procedures withfluorescent-tags.

[0177] Genetic typing of various varieties of plants based on DNAsequence differences may be achieved by detection of alteration inelectrophoretic mobility of DNA fragments in gels, with or withoutdenaturing agents. Small sequence deletions and insertions can bevisualized by high resolution gel electrophoresis. DNA fragments ofdifferent sequences may be distinguished on denaturing formamidegradient gels in which the mobilities of different DNA fragments areretarded in the gel at different positions according to their specificmelting or partial melting temperatures (see, e.g., Myers et al.,Science, 230:1242 (1985)).

[0178] Sequence changes at specific locations also may be revealed bynuclease protection assays, such as RNase and S1 protection or thechemical cleavage method (e.g., Cotton et al., Proc. Nat'l. Acad. Sci.,(USA), 85:4397-4401 (1985)).

[0179] Thus, the detection of a specific DNA sequence may be achieved bymethods such as hybridization, RNase protection, chemical cleavage,direct DNA sequencing or the use of restriction enzymes, (e.g.,restriction fragment length polymorphisms (“RFLP”) and Southern blottingof genomic DNA.

[0180] In addition to more conventional gel-electrophoresis and DNAsequencing, mutations also can be detected by in situ analysis.

[0181] A mutation may be ascertained for example, by a DNA sequencingassay. Samples are processed by methods known in the art to capture theRNA. First strand cDNA is synthesized from the RNA samples by adding anoligonucleotide primer consisting of sequences which hybridize to aregion on the mRNA. Reverse transcriptase and deoxynucleotides are addedto allow synthesis of the first strand cDNA. Primer sequences aresynthesized based on the DNA sequences of the phytate biosyntheticenzymes of the invention. The primer sequence is generally comprised ofat least 15 consecutive bases, and may contain at least 30 or even 50consecutive bases.

[0182] Cells carrying mutations or polymorphisms in the gene of thepresent invention may also be detected at the DNA level by a variety oftechniques. The DNA may be used directly for detection or may beamplified enzymatically by using PCR (Saiki et al., Nature, 324:163-166(1986)) prior to analysis. RT-PCR can also be used to detect mutations.It is particularly preferred to used RT-PCR in conjunction withautomated detection systems, such as, for example, GeneScan. RNA or cDNAmay also be used for the same purpose, PCR or RT-PCR. As an example, PCRprimers complementary to the nucleic acid encoding phytate biosyntheticenzymes can be used to identify and analyze mutations. Examples ofrepresentative primers are shown below in Table 1. For example,deletions and insertions can be detected by a change in size of theamplified product in comparison to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to radiolabeled RNA oralternatively, radiolabeled antisense DNA sequences. While perfectlymatched sequences can be distinguished from mismatched duplexes by RNaseA digestion or by differences in melting temperatures, preferably pointmutations are identified by sequence analysis. Primers used fordetection of mutations or polymorphisms in myo-inositol 1-phosphatesynthase gene 5′CTCGCTACCTCGCTTCGCATTCCATT3′5′ACGCCACTTGGCTCACTTGTACTCCA3′

[0183] Primers used for detection of mutations or polymorphisms inmyo-inositol monophosphatase-3 gene 5′ACGAGGTTGCGGGCGAACCGAAAAT3′5′TAGGGACCGTTGCCTCAACCTAT3′

[0184] Primers used for detection of mutations or polymorphisms inmyo-inositol 1,3,4-trisphosphate 5/6-kinase gene5′TTCTCTCGGTCGCCGCTACTGG3′ 5′AGCATGAACAGTTAGCACCT3′

[0185] Primers used for detection of mutations or polymorphisms inphosphatidylinositol 3-kinase gene 5′CCGCTTCTCC TCACCTTCCT CT 3′5′TGGCTTGTGA CAGTCAGCAT GT 3′

[0186] The above primers may be used for amplifying phytate biosyntheticenzyme cDNA or genomic clones isolated from a sample derived from anindividual plant. The invention also provides the primers above with 1,2, 3 or 4 nucleotides removed from the 5′ and/or the 3′ end. The primersmay be used to amplify the gene isolated from the individual such thatthe gene may then be subject to various techniques for elucidation ofthe DNA sequence. In this way, mutations in the DNA sequence may beidentified.

[0187] Polypeptide Assays

[0188] The present invention also relates to diagnostic assays such asquantitative and diagnostic assays for detecting levels of phytatebiosynthetic enzymes in cells and tissues, including determination ofnormal and abnormal levels. Thus, for instance, a diagnostic assay inaccordance with the invention for detecting expression of phytatebiosynthetic enzymes compared to normal control tissue samples may beused to detect unacceptable levels of expression. Assay techniques thatcan be used to determine levels of polypeptides of the presentinvention, in a sample derived from a plant source are well-known tothose of skill in the art. Such assay methods include radioimmunoassays,competitive-binding assays, Western Blot analysis and ELISA assays.Among these ELISAs frequently are preferred. An ELISA assay initiallycomprises preparing an antibody specific to the polypeptide, preferablya monoclonal antibody. In addition a reporter antibody generally isprepared which binds to the monoclonal antibody. The reporter antibodyis attached to a detectable reagent such as radioactive, fluorescent orenzymatic reagent, in this example horseradish peroxidase enzyme.

[0189] To carry out an ELISA a sample is removed from a host andincubated on a solid support, e.g., a polystyrene dish, that binds theproteins in the sample. Any free protein binding sites on the dish arethen covered by incubating with a non-specific protein such as bovineserum albumin. Next, the monoclonal antibody is incubated in the dishduring which time the monoclonal antibodies attach to any phytatebiosynthetic enzymes attached to the polystyrene dish. Unboundmonoclonal antibody is washed out with buffer. The reporter antibodylinked to horseradish peroxidase is placed in the dish resulting inbinding of the reporter antibody to any monoclonal antibody bound tophytate biosynthetic enzyme. Unattached reporter antibody is then washedout. Reagents for peroxidase activity, including a colorimetricsubstrate are then added to the dish. Immobilized peroxidase, linked tophytate biosynthetic enzyme through the primary and secondaryantibodies, produces a colored reaction product. The amount of colordeveloped in a given time period indicates the amount of phytatebiosynthetic enzyme present in the sample. Quantitative resultstypically are obtained by reference to a standard curve.

[0190] A competition assay may be employed wherein antibodies specificto phytate biosynthetic enzymes attached to a solid support and labeledenzyme derived from the host are passed over the solid support and theamount of label detected attached to the solid support can be correlatedto a quantity of phytate biosynthetic enzyme in the sample.

[0191] Antibodies

[0192] The polypeptides, their fragments or other derivatives, oranalogs thereof, or cells expressing them can be used as immunogens toproduce antibodies thereto. These antibodies can be, for example,polyclonal or monoclonal antibodies. The present invention also includeschimeric, single chain, and humanized antibodies, as well as Fabfragments, or the product of an Fab expression library. Variousprocedures known in the art may be used for the production of suchantibodies and fragments.

[0193] Antibodies generated against the polypeptides corresponding to asequence of the present invention can be obtained by direct injection ofthe polypeptides into an animal or by administering the polypeptides toan animal, preferably a nonhuman. The antibody so obtained will thenbind the polypeptides itself. In this manner, even a sequence encodingonly a fragment of the polypeptide can be used to generate antibodiesbinding the whole native polypeptide. Such antibodies can then be usedto isolate the polypeptide from tissue expressing that polypeptide.

[0194] For preparation of monoclonal antibodies, any technique whichprovides antibodies produced by continuous cell line cultures can beused. Examples include the hybridoma technique (Kohler, G. and Milstein,C., Nature 256:495-497 (1975)), the trioma technique, the human B-cellhybridoma technique (Kozbor et al., Immunology Today 4:72 (1983)) andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., pg. 77-96 in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc. (1985)).

[0195] Hybridoma cell lines secreting the monoclonal antibody areanother aspect of this invention.

[0196] Techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778) can be adapted to produce singlechain antibodies to immunogenic polypeptide products of this invention.Also, transgenic mice, or other organisms such as other mammals, may beused to express humanized antibodies to immunogenic polypeptide productsof this invention.

[0197] The above-described antibodies may be employed to isolate or toidentify clones expressing the polypeptide or purify the polypeptide ofthe present invention by attachment of the antibody to a solid supportfor isolation and/or purification by affinity chromatography.

[0198] Polypeptide derivatives include antigenically or immunologicallyequivalent derivatives which form a particular aspect of this invention.

[0199] The term ‘antigenically equivalent derivative’ as used hereinencompasses a polypeptide or its equivalent which will be specificallyrecognized by certain antibodies which, when raised to the protein orpolypeptide according to the present invention, interfere with theimmediate physical interaction between the antibody and its cognateantigen.

[0200] The term “immunologically equivalent derivative” as used hereinencompasses a peptide or its equivalent which when used in a suitableformulation to raise antibodies in a vertebrate, the antibodies act tointerfere with the immediate physical interaction between the antibodyand its cognate antigen The polypeptide, such as an antigenically orimmunologically equivalent derivative or a fusion protein thereof isused as an antigen to immunize a mouse or other animal such as a ratguinea pig, goat, rabbit, sheep, cattle or chicken. The fusion proteinmay provide stability to the polypeptide. The antigen may be associated,for example by conjugation, with an immunogenic carrier protein forexample bovine serum albumin (BSA) or keyhole limpet haemocyanin (KLH).Alternatively a multiple antigenic peptide comprising multiple copies ofthe protein or polypeptide, or an antigenically or immunologicallyequivalent polypeptide thereof may be sufficiently antigenic to improveimmunogenicity so as to obviate the use of a carrier.

[0201] Alternatively phage display technology could be utilized toselect antibody genes with binding activities towards the polypeptideeither from repertoires of PCR amplified v-genes of lymphocytes fromhumans screened for possessing anti-Fbp or from naive libraries(McCafferty, J. et al., (1990), Nature 348:552-554; Marks, J. et al.,(1992) Biotechnology 10:779-783). The affinity of these antibodies canalso be improved by chain shuffling (Clackson, T. et al., (1991) Nature352:624-628).

[0202] The antibody should be screened again for high affinity to thepolypeptide and/or fusion protein.

[0203] As mentioned above, a fragment of the final antibody may beprepared.

[0204] The antibody may be either intact antibody of M_(r) approximately150,000 or a derivative of it, for example a Fab fragment or a Fvfragment as described in Sierra, A and Pluckthun, A., Science240:1038-1040 (1988). If two antigen binding domains are present eachdomain may be directed against a different epitope—termed ‘bispecific’antibodies.

[0205] The antibody of the invention, as mentioned above, may beprepared by conventional means for example by established monoclonalantibody technology (Kohler, G. and Milstein, C., Nature, 256:495-497(1975)) or using recombinant means e.g. combinatorial libraries, forexample as described in Huse, W. D. et al., Science 246:1275-1281(1989).

[0206] Preferably the antibody is prepared by expression of a DNApolymer encoding said antibody in an appropriate expression system suchas described above for the expression of polypeptides of the invention.The choice of vector for the expression system will be determined inpart by the host, which may be a prokaryotic cell, such as E. coli(preferably strain B) or Streptomyces sp. or a eukaryotic cell, such asa mouse Cl 27, mouse myeloma, human HeLa, Chinese hamster ovary,filamentous or unicellular fungi or insect cell. The host may also be atransgenic animal or a transgenic plant for example as described inHiatt, A. et al., Nature 340:76-78 (1989). Suitable vectors includeplasmids, bacteriophages, cosmids and recombinant viruses, derived from,for example, baculoviruses and vaccinia.

[0207] The Fab fragment may also be prepared from its parent monoclonalantibody by enzyme treatment, for example using papain to cleave the Fabportion from the Fc portion.

[0208] Phytate Biosynthetic Enzyme Binding Molecules and Assays

[0209] This invention also provides a method for identification ofmolecules, such as binding molecules, that bind the phytate biosyntheticenzymes. Genes encoding proteins that bind the enzymes, such as bindingproteins, can be identified by numerous methods known to those of skillin the art, for example, ligand panning and FACS sorting. Such methodsare described in many laboratory manuals such as, for instance, Coliganet al., Current Protocols in Immunology 1(2): Chapter 5 (1991).

[0210] For instance, expression cloning may be employed for thispurpose. To this end polyadenylated RNA is prepared from a cellexpressing the phytate biosynthetic enzymes, a cDNA library is createdfrom this RNA, the library is divided into pools and the pools aretransfected individually into cells that are not expressing the enzyme.The transfected cells then are exposed to labeled enzyme. The enzyme canbe labeled by a variety of well-known techniques including standardmethods of radio-iodination or inclusion of a recognition site for asite-specific protein kinase. Following exposure, the cells are fixedand binding of enzyme is determined. These procedures conveniently arecarried out on glass slides.

[0211] Pools are identified of cDNA that produced phytatebiosyntheticenzyme-binding cells. Sub-pools are prepared from thesepositives, transfected into host cells and screened as described above.Using an iterative sub-pooling and re-screening process, one or moresingle clones that encode the putative binding molecule can be isolated.

[0212] Alternatively a labeled ligand can be photoaffinity linked to acell extract, such as a membrane or a membrane extract, prepared fromcells that express a molecule that it binds, such as a binding molecule.Cross-linked material is resolved by polyacrylamide gel electrophoresis(“PAGE”) and exposed to X-ray film. The labeled complex containing theligand-binding can be excised, resolved into peptide fragments, andsubjected to protein microsequencing. The amino acid sequence obtainedfrom microsequencing can be used to design unique or degenerateoligonucleotide probes to screen cDNA libraries to identify genesencoding the putative binding molecule.

[0213] Polypeptides of the invention also can be used to assess phytatebiosynthetic enzyme binding capacity of phytate biosynthetic enzymebinding molecules, such as binding molecules, in cells or in cell-freepreparations.

[0214] Polypeptides of the invention may also be used to assess thebinding or small molecule substrates and ligands in, for example, cells,cell-free preparations, chemical libraries, and natural productmixtures. These substrates and ligands may be natural substrates andligands or may be structural or functional mimetics.

[0215] Anti-phytate biosynthetic enzyme antibodies represent a usefulclass of binding molecules contemplated by this invention.

[0216] Antagonists—Assays and Molecules

[0217] The invention also provides a method of screening compounds toidentify those which enhance or block the action of phytate biosyntheticenzymes on cells, such as its interaction with substrate molecules. Anantagonist is a compound which decreases the natural biologicalfunctions of the enzymes.

[0218] Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polypeptide of the inventionand thereby inhibit or extinguish its activity. Potential antagonistsalso may be small organic molecules, a peptide, a polypeptide such as aclosely related protein or antibody that binds the same sites on abinding molecule, such as a binding molecule, without inducing phytatebiosynthetic enzyme-induced activities, thereby preventing the action ofthe enzyme by excluding the enzyme from binding.

[0219] Potential antagonists include a small molecule which binds to andoccupies the binding site of the polypeptide thereby preventing bindingto cellular binding molecules, such as binding molecules, such thatnormal biological activity is prevented. Examples of small moleculesinclude but are not limited to small organic molecules, peptides orpeptide-like molecules.

[0220] Other potential antagonists include molecules that affect theexpression of the gene encoding phytate biosynthetic enzymes (e.g.transactivation inhibitors). Other potential antagonists includeantisense molecules. Antisense technology can be used to control geneexpression through antisense DNA or RNA or through double- ortriple-helix formation. Antisense techniques are discussed, for example,in—Okano, J. Neurochem. 56: 560 (1991); OLIGODEOXYNUCLEOTIDES ASANTISENSE INHIBITORS OF GENE EXPRESSION, CRC Press, Boca Raton, Fla.(1988). Triple helix formation is discussed in, for instance Lee et al.,Nucleic Acids Research 6:3073 (1979); Cooney et al., Science 241.456(1988); and Dervan et al., Science 251:1360 (1991). The methods arebased on binding of a polynucleotide to a complementary DNA or RNA. Forexample, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of phytate biosynthetic enzymes. Theantisense RNA oligonucleotide hybridizes to the mRNA in vivo and blockstranslation of the mRNA molecule into phytate biosynthetic enzymes. Theoligonucleotides described above can also be delivered to cells suchthat the antisense RNA or DNA may be expressed in vivo to inhibitproduction of phytate biosynthetic enzymes.

[0221] The antagonists may be employed for instance to reduce the levelsof phytate and/or increase the available phosphorous in plant cells.

EXAMPLES

[0222] The present invention is further described by the followingexamples. The examples are provided solely to illustrate the inventionby reference to specific embodiments. These exemplifications, whileillustrating certain specific aspects of the invention, do not portraythe limitations or circumscribe the scope of the disclosed invention.

[0223] Certain terms used herein are explained in the foregoingglossary.

[0224] All examples were carried out using standard techniques, whichare well known and routine to those of skill in the art, except whereotherwise described in detail. Routine molecular biology techniques ofthe following examples can be carried out as described in standardlaboratory manuals, such as Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989).

[0225] All parts or amounts set out in the following examples are byweight, unless otherwise specified.

[0226] Unless otherwise stated size separation of fragments in theexamples below was carried out using standard techniques of agarose andpolyacrylamide gel electrophoresis (“PAGE”) in Sambrook et al.,MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989) and numerous otherreferences such as, for instance, by Goeddel et al., Nucleic Acids Res.8:4057 (1980).

[0227] Unless described otherwise, ligations were accomplished usingstandard buffers, incubation temperatures and times, approximatelyequimolar amounts of the DNA fragments to be ligated and approximately10 units of T4 DNA ligase (“ligase”) per 0.5 microgram of DNA.

Example 1 Isolation of DNA Coding for Novel Proteins from Zea mays

[0228] The polynucleotide having the myo-inositol 1-phosphate synthaseDNA sequence was obtained from the sequencing of a library of cDNAclones prepared from maize embryos isolated 15 days after pollination.The polynucleotide having the myo-inositol monophosphatase-3 DNAsequence was obtained from the sequencing of a library of cDNA clonesprepared from maize immature ears. The polynucleotide having themyo-inositol 1,3,4-triphosphate 5.6-kinase DNA sequence was obtainedfrom the sequencing of a library of cDNA clones prepared from maizetassel shoots. The polynucleotide having thephosphatidylinositol-3-kinase DNA sequence was obtained from thesequencing of a library of cDNA clones prepared from germinating maizeseeds. Total RNA was isolated from this tissue using standard protocolsand enriched for mRNA by selection with oligo dT, again by standardprotocols. This mRNA was then used as template to synthesizecomplementary DNA (cDNA) using the enzyme reverse transcriptase byconventional methods. The resulting strand of cDNA was then converted todouble-stranded pieces of cDNA and ligated into the cloning vectorpSPORT using conventional ligation/transformation methods. Individualcolonies were then selected and plasmid DNA prepared from each. Thisplasmid DNA was then denatured and used as template in dideoxynucleotidesequencing reactions. By sequencing the individual clones thusidentified with sequencing primers designed from the original sequenceit is then possible to extend the sequence in both directions todetermine the full gene sequence. Suitable techniques are described byManiatis, T., Fritsch, E. F. and Sambrook et al., MOLECULAR CLONING, ALABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989). (See Screening By Hybridization 1.90 andSequencing Denatured Double-Stranded DNA Templates 13.70). The sequenceswere compared to those sequences available in public databases (i.e.,Genbank) to determine homologies/gene identification. In some cases thesequencing data from two or more clones containing overlapping segmentsof DNA were used to construct the contiguous DNA sequence below.

Example 2 Construction of Expression Cassettes for Homology-dependentGene Silencing of Phytate Biosynthetic Enzyme Expression

[0229] To facilitate manipulations of this trait in conventionalbreeding programs, the expression cassette described above is used inhomologous gene silencing (i.e. Knockout) of the endogenous phytatebiosynthetic enzyme polynucleotides by using the embryo-preferredpromoter globulin-1 to drive expression of the genes.

[0230] Plant expression cassettes are made using the embryo-preferredpromoter globulin-1 to drive expression of the phytate biosyntheticenzyme polynucleotides. Globulin-1 termination sequences are alsoincluded in this cassette. The entire expression cassette is cloned intoa pUC based plasmid vector for easy manipulation in E. coli. Thisconstruct is used for particle bombardment transformation of corn inconjunction with another expression construct which includes aselectable marker (for example Pat, PHP8092→Ubi::mo-PAT::ubi). ForAgrobacterium-mediated transformation, a plasmid is moved into anappropriate binary vector containing both left and right bordersequences to facilitate DNA transfer into the target genome.

[0231] This polynucleotide, encoding the inventive polypeptides, whenmade to be non-functional in plants, results in a reduction in phyticacid and an increase in non-phytate phosphorus levels. This can bedemonstrated using the transposable element Mu. Maize lines areconfirmed as having a Mu element inserted into the coding region of thephytate biosynthetic enzyme polynucleotides. Extensive genetics are doneon this phenotype demonstrating it to be transmitted to progeny as ahomozygous recessive trait.

Example 3 Transformation of Maize

[0232] The inventive polynucleotides contained within a vector aretransformed into embryogenic maize callus by particle bombardment.Transgenic maize plants are produced by bombardment of embryogenicallyresponsive immature embryos with tungsten particles associated with DNAplasmids. The plasmids consist of a selectable and an unselectablemarker gene.

[0233] Preparation of Particles

[0234] Fifteen mg of tungsten particles (General Electric), 0.5 to 1.8μ, preferably 1 to 1.8 μ, and most preferably 1 μ, are added to 2 ml ofconcentrated nitric acid. This suspension was sonicated at 0° C. for 20minutes (Branson Sonifier Model 450, 40% output, constant duty cycle).Tungsten particles are pelleted by centrifugation at 10000 rpm (Biofuge)for one minute, and the supernatant is removed. Two milliliters ofsterile distilled water are added to the pellet, and brief sonication isused to resuspend the particles. The suspension is pelleted, onemilliliter of absolute ethanol is added to the pellet, and briefsonication is used to resuspend the particles. Rinsing, pelleting, andresuspending of the particles is performed two more times with steriledistilled water, and finally the particles are resuspended in twomilliliters of sterile distilled water. The particles are subdividedinto 250-ml aliquots and stored frozen.

[0235] Preparation of Particle-plasmid DNA Association

[0236] The stock of tungsten particles are sonicated briefly in a waterbath sonicator (Branson Sonifier Model 450, 20% output, constant dutycycle) and 50 ml is transferred to a microfuge tube. Equimolar amountsof selectable and unselectable plasmid DNA are added to the particlesfor a final DNA amount of 0.1 to 10 mg in 10 ml total volume, andbriefly sonicated. Preferably, 1 mg total DNA is used. Specifically, 4.9ml of PHP 8092 (Ubiquitin::ubiquitinintron::mo-PAT::35SCaMV, 6.329 kbp))plus 5.1 ml of (globulin 1::mi1ps::globulinl),where any phytatebiosyntheticenzyme polynucleotide can replace mi1ps, both at 0.1 mg/mlin TE buffer, are added to the particle suspension. Fifty microliters ofsterile aqueous 2.5 M CaCl₂ are added, and the mixture is brieflysonicated and vortexed. Twenty microliters of sterile aqueous 0.1 Mspermidine are added and the mixture is briefly sonicated and vortexed.The mixture is incubated at room temperature for 20 minutes withintermittent brief sonication. The particle suspension is centrifuged,and the supernatant is removed. Two hundred fifty microliters ofabsolute ethanol are added to the pellet, followed by brief sonication.The suspension is pelleted, the supernatant is removed, and 60 ml ofabsolute ethanol are added. The suspension is sonicated briefly beforeloading the particle-DNA agglomeration onto macrocarriers.

[0237] Preparation of Tissue

[0238] Immature embryos of maize variety High Type II are the target forparticle bombardment-mediated transformation. This genotype is the F₁ oftwo purebred genetic lines, parents A and B, derived from the cross oftwo know maize inbreds, A188 and B73. Both parents are selected for highcompetence of somatic embryogenesis, according to Armstrong et al.,Maize Genetics Coop. News 65:92 (1991).

[0239] Ears from F₁ plants are selfed or sibbed, and embryos areaseptically dissected from developing caryopses when the scutellum firstbecame opaque. This stage occurs about 9-13 days post-pollination, andmost generally about 10 days post-pollination, depending on growthconditions. The embryos are about 0.75 to 1.5 millimeters long. Ears aresurface sterilized with 20-50% Clorox for 30 minutes, followed by threerinses with sterile distilled water.

[0240] Immature embryos are cultured with the scutellum oriented upward,on embryogenic induction medium comprised of N6 basal salts, Erikssonvitamins, 0.5 mg/l thiamine HCl, 30 gm/l sucrose, 2.88 gm/l L-proline, 1mg/l 2,4-dichlorophenoxyacetic acid, 2 gm/l Gelrite, and 8.5 mg/l AgNO₃.Chu et al., Sci. Sin. 18:659 (1975); Eriksson, Physiol. Plant 18:976(1965). The medium is sterilized by autoclaving at 121° C. for 15minutes and dispensed into 100×25 mm Petri dishes. AgNO₃ isfilter-sterilized and added to the medium after autoclaving. The tissuesare cultured in complete darkness at 28° C. After about 3 to 7 days,most usually about 4 days, the scutellum of the embryo swells to aboutdouble its original size and the protuberances at the coleorhizalsurface of the scutellum indicated the inception of embryogenic tissue.Up to 100% of the embryos displayed this response, but most commonly,the embryogenic response frequency is about 80%.

[0241] When the embryogenic response is observed, the embryos aretransferred to a medium comprised of induction medium modified tocontain 120 gm/l sucrose. The embryos are oriented with the coleorhizalpole, the embryogenically responsive tissue, upwards from the culturemedium. Ten embryos per Petri dish are located in the center of a Petridish in an area about 2 cm in diameter. The embryos are maintained onthis medium for 3-16 hour, preferably 4 hours, in complete darkness at28° C. just prior to bombardment with particles associated with plasmidDNAs containing the selectable and unselectable marker genes.

[0242] To effect particle bombardment of embryos, the particle-DNAagglomerates are accelerated using a DuPont PDS-1000 particleacceleration device. The particle-DNA agglomeration is briefly sonicatedand 10 ml were deposited on macrocarriers and the ethanol is allowed toevaporate. The macrocarrieris accelerated onto a stainless-steelstopping screen by the rupture of a polymer diaphragm (rupture disk).Rupture is effected by pressurized helium. The velocity of particle-DNAacceleration is determined based on the rupture disk breaking pressure.Rupture disk pressures of 200 to 1800 psi are used, with 650 to 1100 psibeing preferred, and about 900 psi being most highly preferred. Multipledisks are used to effect a range of rupture pressures.

[0243] The shelf containing the plate with embryos is placed 5.1 cmbelow the bottom of the macrocarrier platform (shelf #3). To effectparticle bombardment of cultured immature embryos, a rupture disk and amacrocarrier with dried particle-DNA agglomerates are installed in thedevice. The He pressure delivered to the device is adjusted to 200 psiabove the rupture disk breaking pressure. A Petri dish with the targetembryos is placed into the vacuum chamber and located in the projectedpath of accelerated particles. A vacuum is created in the chamber,preferably about 28 in Hg. After operation of the device, the vacuum isreleased and the Petri dish is removed.

[0244] Bombarded embryos remain on the osmotically-adjusted mediumduring bombardment, and 1 to 4 days subsequently. The embryos aretransferred to selection medium comprised of N6 basal salts, Erikssonvitamins, 0.5 mg/1 thiamine HCl, 30 gm/l sucrose, 1 mg/l2,4-dichlorophenoxyacetic acid, 2 gm/l Gelrite, 0.85 mg/l Ag NO₃ and 3mg/l bialaphos (Herbiace, Meiji). Bialaphos is added filter-sterilized.The embryos are subcultured to fresh selection medium at 10 to 14 dayintervals. After about 7 weeks, embryogenic tissue, putativelytransformed for both selectable and unselected marker genes,proliferates from about 7% of the bombarded embryos. Putative transgenictissue is rescued, and that tissue derived from individual embryos isconsidered to be an event and is propagated independently on selectionmedium. Two cycles of clonal propagation are achieved by visualselection for the smallest contiguous fragments of organized embryogenictissue.

[0245] A sample of tissue from each event is processed to recover DNA.The DNA is restricted with a restriction endonuclease and probed withprimer sequences designed to amplify DNA sequences overlapping thephytate biosynthetic enzymes and non-phytate biosynthetic enzyme portionof the plasmid. Embryogenic tissue with amplifiable sequence is advancedto plant regeneration.

[0246] For regeneration of transgenic plants, embryogenic tissue issubcultured to a medium comprising MS salts and vitamins (Murashige &Skoog, Physiol. Plant 15:473 (1962)), 100 mg/l myo-inositol, 60 gm/lsucrose, 3 gm/l Gelrite, 0.5 mg/l zeatin, 1 mg/l indole-3-acetic acid,26.4 ng/l cis-trans-abscissic acid, and 3 mg/l bialaphos in 100×25 mmPetri dishes, and is incubated in darkness at 28° C. until thedevelopment of well-formed, matured somatic embryos can be seen. Thisrequires about 14 days. Well-formed somatic embryos are opaque andcream-colored, and are comprised of an identifiable scutellum andcoleoptile. The embryos are individually subcultured to a germinationmedium comprising MS salts and vitamins, 100 mg/l myo-inositol, 40 gm/lsucrose and 1.5 gm/l Gelrite in 100×25 mm Petri dishes and incubatedunder a 16 hour light:8 hour dark photoperiod and 40 meinsteinsm⁻²sec⁻¹from cool-white fluorescent tubes. After about 7 days, the somaticembryos have germinated and produced a well-defined shoot and root. Theindividual plants are subcultured to germination medium in 125×25 mmglass tubes to allow further plant development. The plants aremaintained under a 16 hour light:8 hour dark photoperiod and 40meinsteinsm⁻²sec⁻¹ from cool-white fluorescent tubes. After about 7days, the plants are well-established and are transplanted tohorticultural soil, hardened off, and potted into commercial greenhousesoil mixture and grown to sexual maturity in a greenhouse. An eliteinbred line is used as a male to pollinate regenerated transgenicplants.

Example 4 Identification of High Phosphorus Transgenic Corn Lines

[0247] The resulting transformants are screened for elevated levels ofinorganic phosphorus using a simple calorimetric assay. Individualtransgenic kernels are crushed in the well of a megatiter breeding trayusing a hydraulic press to 2000 psi. The crushed kernels are then soakedin 2 ml of 1 N H2SO4 for 2 hours at room temperature. Color developmentis then initiated by the addition of 4 ml of developing solution (1 part10% ascorbic acid, 6 parts 0.42% ammonium molybdate in 1N H2SO4) to eachcrushed kernel. Kernels are scored after 30 minute incubation at roomtemperature as either positive (blue) or negative (clear). Positive inthis instance refers to a high level of inorganic phosphorus. Thisprotocol is a modified version of what is described in Chen et al.,Anal. Chem. 28:1756 (1956). Those transformants which are screened aspositive with the colorimetric assay will then be subjected to morerigorous analyses to include Southern, Northern and Western blotting andquantitation of phytic acid levels.

[0248] Confirmation of Elevated Non-phytate Phosphorus Levels

[0249] The present transgenics preferably have non-phytate phosphoruslevels in excessive of the natural levels of available phosphorus forthe plant species of interest. In respect to corn it is preferred tohave non-phytate phosphorus levels of about 0.175%, more preferablyabout 0.2% and most preferably about 0.225% or higher. These percentagesbeing base on %wt/wt at a 13% moisture basis for both corn seed. Withrespect to soybeans, it is preferred to have non-phytate phosphoruslevels of about 0.47%, more preferably about 0.49% and most preferablyabout 0.51%. These latter percentage being based on the weight ofnon-phytate phosphorus/(non phytate P/gram of meal on a 13% moisturebasis).

[0250] Each plant identified as a potential high phosphorus transgenicis tested again to confirm the original elevated phosphorus reading.Some putative transgenics may not confirm for the elevated phosphorustrait. Those which confirm are selected on the basis of uniformity forthe elevated phosphorus trait.

[0251] Confirmation of Reduced Phytate Levels

[0252] To determine whether high non-phytate phosphorus transgenics arealso characterizes by reduced levels of phytate, the following method isused to quantify the level of phytic acid in a tested sample.

[0253] The sample is ground, placed in a conical plastic centrifuge tubeand treated with hydrochloric acid. It is homogenized with polytron, andextracted at room 25 temperature with vortexing. The extracted sample isplaced in a clinical centrifuge at 2500 RPM for 15 minutes. 2.5 ml ofthe supernatant is removed and added to 25 ml water. The sample iswashed through a SAX® column. The column is washed with HCl, eluted andevaporated to dryness. The dried sample is resuspended in water andfiltered through a 0.45 micrometer syringe tip filter into a vial. 10 to20 microliters of samples are prepared to inject into an HPLC column.

[0254] The eluting solvent is prepared by mixing 515 ml of methanol, 485ml of double distilled water, 8 ml tetrabutyl ammonium hydroxide 40%(TBAH), 200 microliters of 10 N, (5 M) sulfuric acid, 0.5 ml formic acidand 1-3 mg phytic acid. Phytic acid is prepared by placing 16 mg ofsodium phytate in 5 ml of water. This solution is placed on Dowex ionexchange resin (1 ml Dowex-50 acid form on glass wool in 5 ml pipeftetip). This is rinsed with 1-2 ml water, and the filtrate brought to 10ml with water. Concentration is 1 mg/ml phytic acid. 2 ml is used for 1liter of solvent. pH of the solvent is adjusted to 4.10+/−0.05 with 10 Nsulfuric acid. Chromatography is accomplished by pumping the samplethrough a Hamilton PRP-1 reverse phase HPLC column heated to 40 degreescentigrade at a rate of 1 ml per minute. The detection of inositolphosphate is accomplished with a refractive index detector (Waters),which is auto-zeroed at least two (2) minutes before each run.

[0255] The confirmed high phosphorus transgenics are tested in thismanner. Some, but not all, of the mutants evaluated in this way areconfirmed to be low in phytate.

[0256] Sequence Description

[0257] SEQ ID NO:1 PHOSPHATIDYLINOSITOL-3-KINASE cDNA

[0258] SEQ ID NO:2 PHOSPHATIDYLINOSITOL-3-KINASE POLYPEPTIDE

[0259] SEQ ID NO:3 PHOSPHATIDYLINOSITOL-3-KINASE PRIMER

[0260] SEQ ID NO:4 PHOSPHATIDYLINOSITOL-3-KINASE PRIMER

[0261] SEQ ID NO:5 MYO-INOSITOL 1,3,4-TRIPHOSPHATE 5/6-KINASE cDNA

[0262] SEQ ID NO:6 MYO-INOSITOL 1,3,4-TRIPHOSPHATE 5/6-KINASEPOLYPEPTIDE

[0263] SEQ ID NO:7 MYO-INOSITOL 1,3,4-TRIPHOSPHATE 5/6-KINASE GENERIC

[0264] SEQ ID NO:8 MYO-INOSITOL 1,3,4-TRIPHOSPHATE 5/6-KINASE PRIMER

[0265] SEQ ID NO:9 MYO-INOSITOL 1,3,4-TRIPHOSPHATE 5/6-KINASE PRIMER

[0266] SEQ ID NO:10 MYO-INOSITOL 1-PHOSPHATE SYNTHASE cDNA

[0267] SEQ ID NO:11 MYO-INOSITOL 1-PHOSPHATE SYNTHASE POLYPEPTIDE

[0268] SEQ ID NO:12 MYO-INOSITOL 1-PHOSPHATE SYNTHASE PRIMER

[0269] SEQ ID NO:13 MYO-INOSITOL 1-PHOSPHATE SYNTHASE PRIMER

[0270] SEQ ID NO:14 MYO-INOSITOL 1-PHOSPHATE SYNTHASE GENOMIC

[0271] SEQ ID NO:15 MYO-INOSITOL 1-PHOSPHATE SYNTHASE GENOMIC

[0272] SEQ ID NO:16 MYO-INOSITOL MONOPHOSPHATE-3 cDNA

[0273] SEQ ID NO:17 MYO-INOSITOL MONOPHOSPHATE-3 POLYPEPTIDE

[0274] SEQ ID NO:18 MYO-INOSITOL MONOPHOSPHATE-3 PRIMER

[0275] SEQ ID NO:19 MYO-INOSITOL MONOPHOSPHATE-3 PRIMER

[0276] SEQ ID NO:20 INOSITOL POLYPHOSPHATE 5-PHOSPHATASE cDNA

[0277] SEQ ID NO:21 D-MYO-INOSITOL-3-PHOSPHATE SYNTHASE cDNA

[0278] SEQ ID NO:22 1D-MYO-INOSITOL TRIPHOSPHATE 3-KINASE B cDNA

[0279] SEQ ID NO:23 MYO-INOSITOL TRANSPORTER cDNA

[0280] SEQ ID NO:24 MAIZE PHYTASE cDNA

[0281] SEQ ID NO:25 PHOSPHATIDYLINOSITOL TRANSFER PROTEIN cDNA

[0282] SEQ ID NO:26 PHOSPHATIDYLOINOSITOL-4-PHOSPHATE-5-KINASE cDNA

[0283] SEQ ID NO:27 PHOSPHATIDYLINOSITOL-SPECIFIC PHOSPHOLIPASE C cDNA

[0284] SEQ ID NO:28 MYO-INOSITOL MONOPHOSPHATASE-1 cDNA

[0285] SEQ ID NO:29 PHOSPHATIDYLINOSITOL 4-KINASE cDNA

[0286] SEQ ID NO:30 PHOSPHATIDYLINOSITOL (4,5) BISPHOSPHATE5-PHOSPHATASE

[0287] SEQ ID NO: 31 PHOSPHATIDYLINOSITOL SYNTHASE cDNA

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 31 <210> SEQ ID NO 1<211> LENGTH: 3252 <212> TYPE: DNA <213> ORGANISM: Zea mays <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (258)...(2666) <221>NAME/KEY: misc_feature <222> LOCATION: (1)...(3252) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 1 gtcgacccac gcgtccgctcgccgcgggag tcacgcaacc gccgtctcct cgccggcacg 60 cttcgccgcc gccgcctctctcctcctcgt ctcaaccgcc gcctgcacac gcagaaaagg 120 agggagaata agaggatcagcaaaccccaa gccctccact cgtcgccccc tgctgcaatc 180 gccccacccg cctccgcccgccgccgcttc tcctcacctt cctctcccgc gacatctcag 240 ttcttcatca ccaaaag atggtc ggc ggc ggc aac gag ttc cgt ttc ttc 290 Met Val Gly Gly Gly Asn GluPhe Arg Phe Phe 1 5 10 ttg tcc tgc gac atc agc cac ccg ctt gcc ttc cgtgtt ctc cac gca 338 Leu Ser Cys Asp Ile Ser His Pro Leu Ala Phe Arg ValLeu His Ala 15 20 25 gaa cat atc ttg ttg acc gac caa aaa gtc cca gag ctcttt gtt gag 386 Glu His Ile Leu Leu Thr Asp Gln Lys Val Pro Glu Leu PheVal Glu 30 35 40 tgc aag cta tac atc gat ggg atc caa ttt ggg ttg cct gtaaaa aca 434 Cys Lys Leu Tyr Ile Asp Gly Ile Gln Phe Gly Leu Pro Val LysThr 45 50 55 agg ttg gaa cct tct gga ccg aaa tac tgt tgg aat gag ctc ataaca 482 Arg Leu Glu Pro Ser Gly Pro Lys Tyr Cys Trp Asn Glu Leu Ile Thr60 65 70 75 tta agt acc aaa tac agg gac cta aca tcc ctc tcg cag ctt gcattt 530 Leu Ser Thr Lys Tyr Arg Asp Leu Thr Ser Leu Ser Gln Leu Ala Phe80 85 90 acg gtg tgg gat gtc tca tct ggt gag aac cct gag gtt gtc ggt gga578 Thr Val Trp Asp Val Ser Ser Gly Glu Asn Pro Glu Val Val Gly Gly 95100 105 gcc acc ata ttt ctt ttt aac agc aaa agg cag ctt aaa aca gga aga626 Ala Thr Ile Phe Leu Phe Asn Ser Lys Arg Gln Leu Lys Thr Gly Arg 110115 120 cag aag ctg cgg ctg tgg ccc aca aag gag gca gat gga gga gtc ccc674 Gln Lys Leu Arg Leu Trp Pro Thr Lys Glu Ala Asp Gly Gly Val Pro 125130 135 acc aca act cct ggc aag gtt cct agg aat gag agg ggt gag ata gaa722 Thr Thr Thr Pro Gly Lys Val Pro Arg Asn Glu Arg Gly Glu Ile Glu 140145 150 155 cgt ttg gaa agg ctt gtt aac aag tat gag aga ggg cag ata caacat 770 Arg Leu Glu Arg Leu Val Asn Lys Tyr Glu Arg Gly Gln Ile Gln His160 165 170 gtt gat tgg ctt gat cgt ctt gcc ttc agt gct atg gac aaa gctatg 818 Val Asp Trp Leu Asp Arg Leu Ala Phe Ser Ala Met Asp Lys Ala Met175 180 185 gaa aaa gag tgt gag agg aag gcc aat ttg tac cct agt ctg gttgtt 866 Glu Lys Glu Cys Glu Arg Lys Ala Asn Leu Tyr Pro Ser Leu Val Val190 195 200 gaa ttg tgc agt ttc gaa cat aga att gtc ttc cag gaa tct ggagca 914 Glu Leu Cys Ser Phe Glu His Arg Ile Val Phe Gln Glu Ser Gly Ala205 210 215 aat ttt tat aca ccg gcc cca gta tca tta tca aat gaa ctg gttact 962 Asn Phe Tyr Thr Pro Ala Pro Val Ser Leu Ser Asn Glu Leu Val Thr220 225 230 235 gta tgg gac cct gaa ctt gga aga acc aat cca tct gag cacaag cag 1010 Val Trp Asp Pro Glu Leu Gly Arg Thr Asn Pro Ser Glu His LysGln 240 245 250 tta aag ctt gct aag agc ttg act cgt ggg ata gtt gat agagat cta 1058 Leu Lys Leu Ala Lys Ser Leu Thr Arg Gly Ile Val Asp Arg AspLeu 255 260 265 aaa cca agc tca aat gag aga aag tta cta caa aca att attaag ttt 1106 Lys Pro Ser Ser Asn Glu Arg Lys Leu Leu Gln Thr Ile Ile LysPhe 270 275 280 cct cct aca cgc acc ttg gaa gtg gat gag aag caa ttg gtgtgg aag 1154 Pro Pro Thr Arg Thr Leu Glu Val Asp Glu Lys Gln Leu Val TrpLys 285 290 295 ttt cgt ttc tct ttg atg tct gag aag aaa gct cta acg aaattt gtc 1202 Phe Arg Phe Ser Leu Met Ser Glu Lys Lys Ala Leu Thr Lys PheVal 300 305 310 315 cgc tca gtg gat tgg agt gat aac caa gaa gct aag caagct gtt gag 1250 Arg Ser Val Asp Trp Ser Asp Asn Gln Glu Ala Lys Gln AlaVal Glu 320 325 330 ttg att gga aag tgg gaa atg att gat gtg gct gat gcacta gag ctt 1298 Leu Ile Gly Lys Trp Glu Met Ile Asp Val Ala Asp Ala LeuGlu Leu 335 340 345 ctc tca cct gat ttt gaa agc gac gaa gtt cgt ggt tatgct gtc agc 1346 Leu Ser Pro Asp Phe Glu Ser Asp Glu Val Arg Gly Tyr AlaVal Ser 350 355 360 gta ctt gaa agg gct gat gat gaa gaa tta cag tgc tattta ctc cag 1394 Val Leu Glu Arg Ala Asp Asp Glu Glu Leu Gln Cys Tyr LeuLeu Gln 365 370 375 tta gtg caa gct ctt cgg ttt gaa aga tct gac aag tcccgt cta gca 1442 Leu Val Gln Ala Leu Arg Phe Glu Arg Ser Asp Lys Ser ArgLeu Ala 380 385 390 395 ctc ttt ctt gta aac cgt gct ttg tcc aac atc gaaatt gct agc ttc 1490 Leu Phe Leu Val Asn Arg Ala Leu Ser Asn Ile Glu IleAla Ser Phe 400 405 410 ctc cgg tgg tat ata tta gtt gag ctt cac agt cctgca tat gca aga 1538 Leu Arg Trp Tyr Ile Leu Val Glu Leu His Ser Pro AlaTyr Ala Arg 415 420 425 cga tat tat ggc aca tat gac atg ctt gaa aac agtatg atg aaa ttg 1586 Arg Tyr Tyr Gly Thr Tyr Asp Met Leu Glu Asn Ser MetMet Lys Leu 430 435 440 gtt ggt agg gag gat ggg gat gaa gat gga ttt cgactg tgg cag agt 1634 Val Gly Arg Glu Asp Gly Asp Glu Asp Gly Phe Arg LeuTrp Gln Ser 445 450 455 tta acc cgg cag aca gac ctc act gct caa ttg tgttct att atg aag 1682 Leu Thr Arg Gln Thr Asp Leu Thr Ala Gln Leu Cys SerIle Met Lys 460 465 470 475 gat gta aga aat gta aga ggt agc gca caa aagaaa att gaa aaa ttg 1730 Asp Val Arg Asn Val Arg Gly Ser Ala Gln Lys LysIle Glu Lys Leu 480 485 490 agg cag cta tta tca gga gtt ttc agt gag cttaca aac ttt gat gag 1778 Arg Gln Leu Leu Ser Gly Val Phe Ser Glu Leu ThrAsn Phe Asp Glu 495 500 505 cca att cgt tca cca tta gca cca act ctt ctccta aca gga gtt gtg 1826 Pro Ile Arg Ser Pro Leu Ala Pro Thr Leu Leu LeuThr Gly Val Val 510 515 520 cct caa gaa tcg tct ata ttt aag agt gcc ttgaac cct ttg cgc ctg 1874 Pro Gln Glu Ser Ser Ile Phe Lys Ser Ala Leu AsnPro Leu Arg Leu 525 530 535 aca ttt aaa aca gca aat ggc gga aca tcc aagatt att tac aaa aag 1922 Thr Phe Lys Thr Ala Asn Gly Gly Thr Ser Lys IleIle Tyr Lys Lys 540 545 550 555 ggt gat gac ctc cgg caa gat cag ttg gttatt caa acg gtt tct ttg 1970 Gly Asp Asp Leu Arg Gln Asp Gln Leu Val IleGln Thr Val Ser Leu 560 565 570 atg gac cga cta ctc aaa tta gaa aat ctagat ttg cac ctt act cca 2018 Met Asp Arg Leu Leu Lys Leu Glu Asn Leu AspLeu His Leu Thr Pro 575 580 585 tac cga gtt ctt gca act gga caa gat gaaggg atg ctt gaa ttt att 2066 Tyr Arg Val Leu Ala Thr Gly Gln Asp Glu GlyMet Leu Glu Phe Ile 590 595 600 agt tcc agt tct ctt gca cag att cta tcagaa cat cgc agt att aca 2114 Ser Ser Ser Ser Leu Ala Gln Ile Leu Ser GluHis Arg Ser Ile Thr 605 610 615 agt tac cta cag aag ttc cat cnt gat gaggat ggt cct ttt ggt ata 2162 Ser Tyr Leu Gln Lys Phe His Xaa Asp Glu AspGly Pro Phe Gly Ile 620 625 630 635 acg gct caa tgt ttg gag aca ttc ataaaa agc tgc gcc ggt tac tct 2210 Thr Ala Gln Cys Leu Glu Thr Phe Ile LysSer Cys Ala Gly Tyr Ser 640 645 650 gtc att aca tac ata ttg ggg gtt ggagac agg cat ctg gat aat ctt 2258 Val Ile Thr Tyr Ile Leu Gly Val Gly AspArg His Leu Asp Asn Leu 655 660 665 ctt cta act gat gat gga cgc ctt tttcat gtt gac ttt gct ttt atc 2306 Leu Leu Thr Asp Asp Gly Arg Leu Phe HisVal Asp Phe Ala Phe Ile 670 675 680 ctt ggg cga gac cca aag cca ttt ccgcca ccg atg aag ttg tgt aag 2354 Leu Gly Arg Asp Pro Lys Pro Phe Pro ProPro Met Lys Leu Cys Lys 685 690 695 gaa atg gtt gag gcc atg ggt ggt gcagaa agc caa tat tac aca agg 2402 Glu Met Val Glu Ala Met Gly Gly Ala GluSer Gln Tyr Tyr Thr Arg 700 705 710 715 ttc aag tcc tac tgc tgc gaa gcatac aac att ctg agg aag tcc agc 2450 Phe Lys Ser Tyr Cys Cys Glu Ala TyrAsn Ile Leu Arg Lys Ser Ser 720 725 730 agt ctc att ttg aat cta ttc aagctg atg gag cga tca ggc att ccg 2498 Ser Leu Ile Leu Asn Leu Phe Lys LeuMet Glu Arg Ser Gly Ile Pro 735 740 745 gac atc tct gcc gat gaa agc ggaggt ctc aag ctc cag gag aaa ttc 2546 Asp Ile Ser Ala Asp Glu Ser Gly GlyLeu Lys Leu Gln Glu Lys Phe 750 755 760 cgg ttg gat ctg gac gac gag gaggct ata cat ttc ttc cag gat ctt 2594 Arg Leu Asp Leu Asp Asp Glu Glu AlaIle His Phe Phe Gln Asp Leu 765 770 775 atc aac gat agc gtg agt gct ctgttc cct caa atg gtt gag acc atc 2642 Ile Asn Asp Ser Val Ser Ala Leu PhePro Gln Met Val Glu Thr Ile 780 785 790 795 cat aga tgg gct caa tat tggcgg taacacaagc taatgtcgta gaagcaagtg 2696 His Arg Trp Ala Gln Tyr TrpArg 800 tgaatctgta catgctgact gtcacaagcc acggtattaa gcgagaaacgacacttgatg 2756 gatggaagct taggcgctta gcatttgggg ttcaagctgc nccgcatctgcgaattgatt 2816 gggctgatgc agggcatggg caatcttctt cgtgccggtg acacccaggaattcgggttg 2876 tcagttgtca cttgtgatag tagaattccg tcacgcactg ctgtagacctatgggcattc 2936 gtcagatgta tatatgcgtt aatgtataaa atcaacttca gtagcaaatttgtgaatacc 2996 ggaatacgtg atggtttagg gcctgtttgt ttaccccatg gattatataatctggattat 3056 ttttggagga ttatataatc tggattatat aatctgagta gttctgtttgtttacccaga 3116 ttatttgagt tgttaatagg attcttttgt atgaggaaga caagaatgccctctatattt 3176 gtactaggtt gaaactcata tatgagatga acaatgtaac aaaaaaaaaaaaaaaaaaaa 3236 aaaaaaaggg cggccg 3252 <210> SEQ ID NO 2 <211> LENGTH:803 <212> TYPE: PRT <213> ORGANISM: Zea mays <220> FEATURE: <221>NAME/KEY: VARIANT <222> LOCATION: (1)...(803) <223> OTHER INFORMATION:Xaa = Any Amino Acid <400> SEQUENCE: 2 Met Val Gly Gly Gly Asn Glu PheArg Phe Phe Leu Ser Cys Asp Ile 1 5 10 15 Ser His Pro Leu Ala Phe ArgVal Leu His Ala Glu His Ile Leu Leu 20 25 30 Thr Asp Gln Lys Val Pro GluLeu Phe Val Glu Cys Lys Leu Tyr Ile 35 40 45 Asp Gly Ile Gln Phe Gly LeuPro Val Lys Thr Arg Leu Glu Pro Ser 50 55 60 Gly Pro Lys Tyr Cys Trp AsnGlu Leu Ile Thr Leu Ser Thr Lys Tyr 65 70 75 80 Arg Asp Leu Thr Ser LeuSer Gln Leu Ala Phe Thr Val Trp Asp Val 85 90 95 Ser Ser Gly Glu Asn ProGlu Val Val Gly Gly Ala Thr Ile Phe Leu 100 105 110 Phe Asn Ser Lys ArgGln Leu Lys Thr Gly Arg Gln Lys Leu Arg Leu 115 120 125 Trp Pro Thr LysGlu Ala Asp Gly Gly Val Pro Thr Thr Thr Pro Gly 130 135 140 Lys Val ProArg Asn Glu Arg Gly Glu Ile Glu Arg Leu Glu Arg Leu 145 150 155 160 ValAsn Lys Tyr Glu Arg Gly Gln Ile Gln His Val Asp Trp Leu Asp 165 170 175Arg Leu Ala Phe Ser Ala Met Asp Lys Ala Met Glu Lys Glu Cys Glu 180 185190 Arg Lys Ala Asn Leu Tyr Pro Ser Leu Val Val Glu Leu Cys Ser Phe 195200 205 Glu His Arg Ile Val Phe Gln Glu Ser Gly Ala Asn Phe Tyr Thr Pro210 215 220 Ala Pro Val Ser Leu Ser Asn Glu Leu Val Thr Val Trp Asp ProGlu 225 230 235 240 Leu Gly Arg Thr Asn Pro Ser Glu His Lys Gln Leu LysLeu Ala Lys 245 250 255 Ser Leu Thr Arg Gly Ile Val Asp Arg Asp Leu LysPro Ser Ser Asn 260 265 270 Glu Arg Lys Leu Leu Gln Thr Ile Ile Lys PhePro Pro Thr Arg Thr 275 280 285 Leu Glu Val Asp Glu Lys Gln Leu Val TrpLys Phe Arg Phe Ser Leu 290 295 300 Met Ser Glu Lys Lys Ala Leu Thr LysPhe Val Arg Ser Val Asp Trp 305 310 315 320 Ser Asp Asn Gln Glu Ala LysGln Ala Val Glu Leu Ile Gly Lys Trp 325 330 335 Glu Met Ile Asp Val AlaAsp Ala Leu Glu Leu Leu Ser Pro Asp Phe 340 345 350 Glu Ser Asp Glu ValArg Gly Tyr Ala Val Ser Val Leu Glu Arg Ala 355 360 365 Asp Asp Glu GluLeu Gln Cys Tyr Leu Leu Gln Leu Val Gln Ala Leu 370 375 380 Arg Phe GluArg Ser Asp Lys Ser Arg Leu Ala Leu Phe Leu Val Asn 385 390 395 400 ArgAla Leu Ser Asn Ile Glu Ile Ala Ser Phe Leu Arg Trp Tyr Ile 405 410 415Leu Val Glu Leu His Ser Pro Ala Tyr Ala Arg Arg Tyr Tyr Gly Thr 420 425430 Tyr Asp Met Leu Glu Asn Ser Met Met Lys Leu Val Gly Arg Glu Asp 435440 445 Gly Asp Glu Asp Gly Phe Arg Leu Trp Gln Ser Leu Thr Arg Gln Thr450 455 460 Asp Leu Thr Ala Gln Leu Cys Ser Ile Met Lys Asp Val Arg AsnVal 465 470 475 480 Arg Gly Ser Ala Gln Lys Lys Ile Glu Lys Leu Arg GlnLeu Leu Ser 485 490 495 Gly Val Phe Ser Glu Leu Thr Asn Phe Asp Glu ProIle Arg Ser Pro 500 505 510 Leu Ala Pro Thr Leu Leu Leu Thr Gly Val ValPro Gln Glu Ser Ser 515 520 525 Ile Phe Lys Ser Ala Leu Asn Pro Leu ArgLeu Thr Phe Lys Thr Ala 530 535 540 Asn Gly Gly Thr Ser Lys Ile Ile TyrLys Lys Gly Asp Asp Leu Arg 545 550 555 560 Gln Asp Gln Leu Val Ile GlnThr Val Ser Leu Met Asp Arg Leu Leu 565 570 575 Lys Leu Glu Asn Leu AspLeu His Leu Thr Pro Tyr Arg Val Leu Ala 580 585 590 Thr Gly Gln Asp GluGly Met Leu Glu Phe Ile Ser Ser Ser Ser Leu 595 600 605 Ala Gln Ile LeuSer Glu His Arg Ser Ile Thr Ser Tyr Leu Gln Lys 610 615 620 Phe His XaaAsp Glu Asp Gly Pro Phe Gly Ile Thr Ala Gln Cys Leu 625 630 635 640 GluThr Phe Ile Lys Ser Cys Ala Gly Tyr Ser Val Ile Thr Tyr Ile 645 650 655Leu Gly Val Gly Asp Arg His Leu Asp Asn Leu Leu Leu Thr Asp Asp 660 665670 Gly Arg Leu Phe His Val Asp Phe Ala Phe Ile Leu Gly Arg Asp Pro 675680 685 Lys Pro Phe Pro Pro Pro Met Lys Leu Cys Lys Glu Met Val Glu Ala690 695 700 Met Gly Gly Ala Glu Ser Gln Tyr Tyr Thr Arg Phe Lys Ser TyrCys 705 710 715 720 Cys Glu Ala Tyr Asn Ile Leu Arg Lys Ser Ser Ser LeuIle Leu Asn 725 730 735 Leu Phe Lys Leu Met Glu Arg Ser Gly Ile Pro AspIle Ser Ala Asp 740 745 750 Glu Ser Gly Gly Leu Lys Leu Gln Glu Lys PheArg Leu Asp Leu Asp 755 760 765 Asp Glu Glu Ala Ile His Phe Phe Gln AspLeu Ile Asn Asp Ser Val 770 775 780 Ser Ala Leu Phe Pro Gln Met Val GluThr Ile His Arg Trp Ala Gln 785 790 795 800 Tyr Trp Arg <210> SEQ ID NO3 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 3ccgcttctcc tcaccttcct ct 22 <210> SEQ ID NO 4 <211> LENGTH: 22 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer <400> SEQUENCE: 4 tggcttgtga cagtcagcat gt 22 <210>SEQ ID NO 5 <211> LENGTH: 1428 <212> TYPE: DNA <213> ORGANISM: Zea mays<220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (118)...(1176) <400>SEQUENCE: 5 cccgggtcga cccacgcgtc cgggtgcccg cccgcacaca ccacctgtccccgctccgct 60 ccgctccgcg ttcccttctc tcggtcgccg ctactggcct tcgctcggtccgccgcg atg 120 Met 1 gtg tct ggc ggg tgc gtg ggg acg gag ggg gag gcggac cgc gcg gcg 168 Val Ser Gly Gly Cys Val Gly Thr Glu Gly Glu Ala AspArg Ala Ala 5 10 15 gcg cct ccg gag gcc gcg gag gag ccg gtg gtg ccg gcgcct ccc gcg 216 Ala Pro Pro Glu Ala Ala Glu Glu Pro Val Val Pro Ala ProPro Ala 20 25 30 cgg gag gtc gtg gtg ggg tac gcg ctc acg acg aag aag gccaag agc 264 Arg Glu Val Val Val Gly Tyr Ala Leu Thr Thr Lys Lys Ala LysSer 35 40 45 ttc ctc cag ccc aag ctc cgg ggg ctc gcc agg aaa aag gga atcttg 312 Phe Leu Gln Pro Lys Leu Arg Gly Leu Ala Arg Lys Lys Gly Ile Leu50 55 60 65 ttt gtt gct att gat cag aaa cgt cca ttg tct gat caa ggt ccattt 360 Phe Val Ala Ile Asp Gln Lys Arg Pro Leu Ser Asp Gln Gly Pro Phe70 75 80 gac att gtt ctt cat aag ttg act gga aag ggg tgg cag caa ttg ctg408 Asp Ile Val Leu His Lys Leu Thr Gly Lys Gly Trp Gln Gln Leu Leu 8590 95 gag gaa tat agg gag gct cac cca gaa gtt act gtt ctt gat cca cct456 Glu Glu Tyr Arg Glu Ala His Pro Glu Val Thr Val Leu Asp Pro Pro 100105 110 ggc gca ata gca aac ttg cta gat cgc caa tct atg ctt caa gaa gta504 Gly Ala Ile Ala Asn Leu Leu Asp Arg Gln Ser Met Leu Gln Glu Val 115120 125 tct gaa ttg gac tca ccg att gtc atg ttc tct tct gca ggt aaa gta552 Ser Glu Leu Asp Ser Pro Ile Val Met Phe Ser Ser Ala Gly Lys Val 130135 140 145 cgc gtg cct aaa cag cta ttc att aat act gat ccc tca tca atacca 600 Arg Val Pro Lys Gln Leu Phe Ile Asn Thr Asp Pro Ser Ser Ile Pro150 155 160 gct gca gtt agg agg gcg ggt ctc tct ctc cca ttg gtg gca aaaccc 648 Ala Ala Val Arg Arg Ala Gly Leu Ser Leu Pro Leu Val Ala Lys Pro165 170 175 ttg gtg gcg aag tcc cat gag cta tcc ctg gct tat gat cca acttca 696 Leu Val Ala Lys Ser His Glu Leu Ser Leu Ala Tyr Asp Pro Thr Ser180 185 190 ctg acc aaa ctt gag ccc cct tta gtt ctt cag gaa ttt gtt aaccat 744 Leu Thr Lys Leu Glu Pro Pro Leu Val Leu Gln Glu Phe Val Asn His195 200 205 gtt ggt gtc atg ttt aag gtg tac att gtt ggg gat gca ata agggtt 792 Val Gly Val Met Phe Lys Val Tyr Ile Val Gly Asp Ala Ile Arg Val210 215 220 225 gta cgt cgg ttt tca ctt cca aat gtt gat gaa ggt gat ctgtcg aat 840 Val Arg Arg Phe Ser Leu Pro Asn Val Asp Glu Gly Asp Leu SerAsn 230 235 240 aat gct ggg gta ttt cgg ttt cca agg gtc tct tgt gct gcagcc agc 888 Asn Ala Gly Val Phe Arg Phe Pro Arg Val Ser Cys Ala Ala AlaSer 245 250 255 gca gat gat gca gat ctt gac cct ggt gtt gct gaa ctt cctccg aga 936 Ala Asp Asp Ala Asp Leu Asp Pro Gly Val Ala Glu Leu Pro ProArg 260 265 270 cca ttg ctt gag atc ttg gca cga gag ctg cgg cga cga ctgggt ctt 984 Pro Leu Leu Glu Ile Leu Ala Arg Glu Leu Arg Arg Arg Leu GlyLeu 275 280 285 aga cta ttc aac att gat atg att agg gag cac gga aca agagac cgg 1032 Arg Leu Phe Asn Ile Asp Met Ile Arg Glu His Gly Thr Arg AspArg 290 295 300 305 ttt tat gtc ata gac atg aac tac ttt cct ggg tac ggcaaa atg ccc 1080 Phe Tyr Val Ile Asp Met Asn Tyr Phe Pro Gly Tyr Gly LysMet Pro 310 315 320 ggg tac gag cac gtg ttc acc gac ttc ctg ctg agc cttgcc cag aaa 1128 Gly Tyr Glu His Val Phe Thr Asp Phe Leu Leu Ser Leu AlaGln Lys 325 330 335 gag tac aag agg cga cca agc tat agc tcc cta ggc tcaggc gaa ggg 1176 Glu Tyr Lys Arg Arg Pro Ser Tyr Ser Ser Leu Gly Ser GlyGlu Gly 340 345 350 tgaaaagtga ggccgaggct actcggcggg ggtgccctgtatatgtctag catccgcaat 1236 gcgtgcgtgc gtgcgtacag atgtgctgcg tgacgggagaggatgggtcg tagagttggg 1296 gcatcactgc atcacatcag tggccgcgat aaaaagaagcgaggactgtt gataggctgt 1356 aattaaattg ttactttgca ggtgctaact gttcatgcttcaaaaaaaaa aaaaaaaaaa 1416 aaagggcggc cg 1428 <210> SEQ ID NO 6 <211>LENGTH: 353 <212> TYPE: PRT <213> ORGANISM: Zea mays <400> SEQUENCE: 6Met Val Ser Gly Gly Cys Val Gly Thr Glu Gly Glu Ala Asp Arg Ala 1 5 1015 Ala Ala Pro Pro Glu Ala Ala Glu Glu Pro Val Val Pro Ala Pro Pro 20 2530 Ala Arg Glu Val Val Val Gly Tyr Ala Leu Thr Thr Lys Lys Ala Lys 35 4045 Ser Phe Leu Gln Pro Lys Leu Arg Gly Leu Ala Arg Lys Lys Gly Ile 50 5560 Leu Phe Val Ala Ile Asp Gln Lys Arg Pro Leu Ser Asp Gln Gly Pro 65 7075 80 Phe Asp Ile Val Leu His Lys Leu Thr Gly Lys Gly Trp Gln Gln Leu 8590 95 Leu Glu Glu Tyr Arg Glu Ala His Pro Glu Val Thr Val Leu Asp Pro100 105 110 Pro Gly Ala Ile Ala Asn Leu Leu Asp Arg Gln Ser Met Leu GlnGlu 115 120 125 Val Ser Glu Leu Asp Ser Pro Ile Val Met Phe Ser Ser AlaGly Lys 130 135 140 Val Arg Val Pro Lys Gln Leu Phe Ile Asn Thr Asp ProSer Ser Ile 145 150 155 160 Pro Ala Ala Val Arg Arg Ala Gly Leu Ser LeuPro Leu Val Ala Lys 165 170 175 Pro Leu Val Ala Lys Ser His Glu Leu SerLeu Ala Tyr Asp Pro Thr 180 185 190 Ser Leu Thr Lys Leu Glu Pro Pro LeuVal Leu Gln Glu Phe Val Asn 195 200 205 His Val Gly Val Met Phe Lys ValTyr Ile Val Gly Asp Ala Ile Arg 210 215 220 Val Val Arg Arg Phe Ser LeuPro Asn Val Asp Glu Gly Asp Leu Ser 225 230 235 240 Asn Asn Ala Gly ValPhe Arg Phe Pro Arg Val Ser Cys Ala Ala Ala 245 250 255 Ser Ala Asp AspAla Asp Leu Asp Pro Gly Val Ala Glu Leu Pro Pro 260 265 270 Arg Pro LeuLeu Glu Ile Leu Ala Arg Glu Leu Arg Arg Arg Leu Gly 275 280 285 Leu ArgLeu Phe Asn Ile Asp Met Ile Arg Glu His Gly Thr Arg Asp 290 295 300 ArgPhe Tyr Val Ile Asp Met Asn Tyr Phe Pro Gly Tyr Gly Lys Met 305 310 315320 Pro Gly Tyr Glu His Val Phe Thr Asp Phe Leu Leu Ser Leu Ala Gln 325330 335 Lys Glu Tyr Lys Arg Arg Pro Ser Tyr Ser Ser Leu Gly Ser Gly Glu340 345 350 Gly <210> SEQ ID NO 7 <211> LENGTH: 1059 <212> TYPE: DNA<213> ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY: misc_feature<222> LOCATION: (1)...(1059) <223> OTHER INFORMATION: n = A,T,C or G<400> SEQUENCE: 7 atggtntcng gnggntgygt nggnacngar ggngargcng aycgngcngcngcnccnccn 60 gargcngcng argarccngt ngtnccngcn ccnccngcnc gngargtngtngtnggntay 120 gcnctnacna cnaaraargc naartcntty ctngarccna arctncgnggnctngcncgn 180 aaraarggna thctnttygt ngcnathgay caraarcgnc cnctntcngaycarggnccn 240 ttygayathg tnctncayaa rctnacnggn aarggntggc arcarctnctngargartay 300 cgngargcnc ayccngargt nacngtnctn gayccnccng gngcnathgcnaayctnctn 360 gaycgncart cnatgctnca rggngtntcn garctngayt cnccnathgtnatgttytcn 420 tcngcnggna argtncgngt nccnaarcar ctnttyatha ayacngayccntcntcnath 480 ccngcngcng tncgncgngc nggnctntcn ctnccnctng tngcnaarccnctngtngcn 540 aartcncayg arctntcnct ngcntaygay ccnacntcnc tnacnaarctngarccnccn 600 ctngtnctnc argarttygt naaycaygtn ggngtnatgt tyaargtntayathgtnggn 660 gaygcnathc gngtngtncg ncgnttytcn ctnccnaayg tngaygarggngayctntcn 720 aayaaygcng gngtnttycg nttyccncgn gtntcntgyg cngcngcntcngcngaygay 780 gcngayctng ayccnggngt ngcngarctn ccnccncgnc cnctnctngarathctngcn 840 cgngarctnc gncgncgnct nggnctncgn ctnttyaaya thgayatgathcgngarcay 900 ggnacncgng aycgnttyta ygtnathgay atgaaytayt tyccnggntayggnaaratg 960 ccnggntayg arcaygtntt yacngaytty ctnctntcnc tngcncaraargartayaar 1020 cgncgnccnt cntaytcntc nctngcntcn ggngarggn 1059 <210>SEQ ID NO 8 <211> LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE:8 ttctctcggt cgccgctact gg 22 <210> SEQ ID NO 9 <211> LENGTH: 20 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer <400> SEQUENCE: 9 agcatgaaca gttagcacct 20 <210> SEQID NO 10 <211> LENGTH: 1931 <212> TYPE: DNA <213> ORGANISM: Zea mays<400> SEQUENCE: 10 ggcacgagca gcagcctcct tcctcctctc actctcgctcgcgctgcgct cgctacctcg 60 cttcgcattc cattcgaaaa gaggggagga aaggcaagatgttcatcgag agcttccgcg 120 tcgagagccc ccacgtgcgg tacggcccga tggagatcgagtcggagtac cggtacgaca 180 cgacggagct ggtacacgag ggcaaggacg gcgcctcacgctgggtcgtc cgccccaagt 240 ccgtcaagta caacttccgg accagaaccg ccgtccccaagctcggggtg atgcttgtgg 300 ggtggggagg caacaacggg tccacgctga cggctggggtcattgccaac agggagggga 360 tctcatgggc gaccaaggac aaggtgcagc aagccaactactacggctcc ctcacccacg 420 cctccaccat cagagtcggc agctacaacg gggaggagatctatgcgccg ttcaagagcc 480 tccttcccat agtgaaccca gacgacattg tgttcggaggctgggacatt agcaacatga 540 acctggccga ctccatgacc agggccaagg tgctggatattgacctgcag aagcagctca 600 ggccctacat ggagtccatg gtgccacttc ccggtatctatgatccggac ttcatcgcgg 660 ctaaccaggg ctctcgcgcc aacagtgtca tcaagggcaccaagaaagaa caggtggagc 720 agatcatcaa ggatatcagg gagtttaagg agaagaacaaagtggacaag atagttgtgt 780 tgtggactgc aaacactgaa aggtatagca atgtgtgcgctggtctcaac gacacgatgg 840 agaatctact ggcatctgtg gacaagaacg aagcggaggtatcaccatca acactatatg 900 ccattgcctg tgtcatggaa ggggtgccgt tcatcaatgggagcccccag aacacctttg 960 tgcctgggct gattgatctt gctataaaaa acaactgcttgattggtggt gacgacttca 1020 agagtggaca gaccaagatg aaatctgtct tggtcgatttccttgttggt gctggaataa 1080 agcccacctc aatcgtgagc tacaaccact tgggaaacaacgatggcatg aacctgtctg 1140 cccctcaaac attcaggtcc aaggagatct ccaagagcaacgtggtggat gacatggtct 1200 cgagcaatgc catcctctat gagcccggcg agcatcccgatcatgtcgtt gtcatcaagt 1260 atgtgccgta cgtgggagac agcaagaggg ctatggacgagtacacctca gagatcttca 1320 tgggcggcaa gaacaccatc gtgctgcaca acacctgtgaggactcgctc ctcgccgcac 1380 ctatcatcct tgatctggtg ctcttggctg agctcagcaccaggatccag ctgaaagctg 1440 agggagagga caaattccac tccttccacc cggtggccaccatcttgagt tacttcacca 1500 aggcacccct ggttccccct ggcacaccgg tggtgaacgctctggccaag cagagggcga 1560 tgctggagaa catcatgagg gcctgcgttg ggctggccccagagaacaac atgatcttgg 1620 agtacaagtg agccaagtgg cgtgccctgc agcgcgaggttagctgctgg aagggaacta 1680 gaaaggcgag attagctgtg ggattgtgtt gggcttgtcgtgttttcttt tgcgttcttt 1740 cctagtcatt gctgttgcgc ttttgtattt gtcggacccgtaactaccag ggctctgcta 1800 ttagcggcac ggagcctgta attgtattgt atgataatgtgatcgagggt gctacttccc 1860 ctcggcattc ctagtgttgg ttaaaagtcg ttcgacagcaacttatcgac ccaaaaaaaa 1920 aaaaaaaaaa a 1931 <210> SEQ ID NO 11 <211>LENGTH: 510 <212> TYPE: PRT <213> ORGANISM: Zea mays <400> SEQUENCE: 11Met Phe Ile Glu Ser Phe Arg Val Glu Ser Pro His Val Arg Tyr Gly 1 5 1015 Pro Met Glu Ile Glu Ser Glu Tyr Arg Tyr Asp Thr Thr Glu Leu Val 20 2530 His Glu Gly Lys Asp Gly Ala Ser Arg Trp Val Val Arg Pro Lys Ser 35 4045 Val Lys Tyr Asn Phe Arg Thr Arg Thr Ala Val Pro Lys Leu Gly Val 50 5560 Met Leu Val Gly Trp Gly Gly Asn Asn Gly Ser Thr Leu Thr Ala Gly 65 7075 80 Val Ile Ala Asn Arg Glu Gly Ile Ser Trp Ala Thr Lys Asp Lys Val 8590 95 Gln Gln Ala Asn Tyr Tyr Gly Ser Leu Thr His Ala Ser Thr Ile Arg100 105 110 Val Gly Ser Tyr Asn Gly Glu Glu Ile Tyr Ala Pro Phe Lys SerLeu 115 120 125 Leu Pro Ile Val Asn Pro Asp Asp Ile Val Phe Gly Gly TrpAsp Ile 130 135 140 Ser Asn Met Asn Leu Ala Asp Ser Met Thr Arg Ala LysVal Leu Asp 145 150 155 160 Ile Asp Leu Gln Lys Gln Leu Arg Pro Tyr MetGlu Ser Met Val Pro 165 170 175 Leu Pro Gly Ile Tyr Asp Pro Asp Phe IleAla Ala Asn Gln Gly Ser 180 185 190 Arg Ala Asn Ser Val Ile Lys Gly ThrLys Lys Glu Gln Val Glu Gln 195 200 205 Ile Ile Lys Asp Ile Arg Glu PheLys Glu Lys Asn Lys Val Asp Lys 210 215 220 Ile Val Val Leu Trp Thr AlaAsn Thr Glu Arg Tyr Ser Asn Val Cys 225 230 235 240 Ala Gly Leu Asn AspThr Met Glu Asn Leu Leu Ala Ser Val Asp Lys 245 250 255 Asn Glu Ala GluVal Ser Pro Ser Thr Leu Tyr Ala Ile Ala Cys Val 260 265 270 Met Glu GlyVal Pro Phe Ile Asn Gly Ser Pro Gln Asn Thr Phe Val 275 280 285 Pro GlyLeu Ile Asp Leu Ala Ile Lys Asn Asn Cys Leu Ile Gly Gly 290 295 300 AspAsp Phe Lys Ser Gly Gln Thr Lys Met Lys Ser Val Leu Val Asp 305 310 315320 Phe Leu Val Gly Ala Gly Ile Lys Pro Thr Ser Ile Val Ser Tyr Asn 325330 335 His Leu Gly Asn Asn Asp Gly Met Asn Leu Ser Ala Pro Gln Thr Phe340 345 350 Arg Ser Lys Glu Ile Ser Lys Ser Asn Val Val Asp Asp Met ValSer 355 360 365 Ser Asn Ala Ile Leu Tyr Glu Pro Gly Glu His Pro Asp HisVal Val 370 375 380 Val Ile Lys Tyr Val Pro Tyr Val Gly Asp Ser Lys ArgAla Met Asp 385 390 395 400 Glu Tyr Thr Ser Glu Ile Phe Met Gly Gly LysAsn Thr Ile Val Leu 405 410 415 His Asn Thr Cys Glu Asp Ser Leu Leu AlaAla Pro Ile Ile Leu Asp 420 425 430 Leu Val Leu Leu Ala Glu Leu Ser ThrArg Ile Gln Leu Lys Ala Glu 435 440 445 Gly Glu Asp Lys Phe His Ser PheHis Pro Val Ala Thr Ile Leu Ser 450 455 460 Tyr Phe Thr Lys Ala Pro LeuVal Pro Pro Gly Thr Pro Val Val Asn 465 470 475 480 Ala Leu Ala Lys GlnArg Ala Met Leu Glu Asn Ile Met Arg Ala Cys 485 490 495 Val Gly Leu AlaPro Glu Asn Asn Met Ile Leu Glu Tyr Lys 500 505 510 <210> SEQ ID NO 12<211> LENGTH: 26 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 12ctcgctacct cgcttcgcat tccatt 26 <210> SEQ ID NO 13 <211> LENGTH: 26<212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: primer <400> SEQUENCE: 13 acgccacttg gctcacttgtactcca 26 <210> SEQ ID NO 14 <211> LENGTH: 3546 <212> TYPE: DNA <213>ORGANISM: Zea mays <400> SEQUENCE: 14 ctcgctacct cgcttcgcat tccattcgaggagagcggtg agaggggagg aaaggcaaga 60 tgttcatcga gagcttccgc gtcgagagcccccacgtgcg gtacggcccg acggagatcg 120 agtcggagta ccggtacgac acgacggagctggtacacga gggcaaggac ggcgcctcac 180 gctgggtcgt ccgccccaag tccgtcaagtacaacttccg gaccagaacc gccgtcccca 240 agctcgggta tgtacggatg cagcggccctagcctcactc tctgtgaacc ctcctcctcc 300 cgtgctcagt caaatcctcc gtcgagatcaactggtcggc gttccctcct aaatcctaat 360 gaaaatctta ctgctttgcc tgaagacgaaccgtcgtaat tgttgacagc tacgcacaca 420 cttgcccatc cggatgcgtc aaatcagctcgatttgaaat tcgattcgat ggtgcccttt 480 tccatatttc gatcatccct cgcctactgtgcaatgatta cagaaacgtc cttttcctct 540 gaactttgtc ttaggctttt tgtcctgtgcacgtgagctg gtatcaattt gttcatgtaa 600 gatcaaattc cagcagggac gatgagcagcagacagaact cattacacta gcaaattgat 660 actaggatta ctggcaagtg tgcatacggcgcaatctgcc atcctggacc ccctttgttt 720 aattcctgtt cctatgcatg ttgcctacgtgcagctcgtt gtgtgttatg gtgtcaggct 780 gtcagccgct tgtctctgtc cgacggatgatgccaacttt tctgttctgg tggtgcaggg 840 tgatgcttgt ggggtgggga ggcaacaacgggtccacgct gacggctggg gtcattgcca 900 acagggagtg agtagtactt aatttgtcctatattgcttt ccgttgtttt cagttattaa 960 tggcctaaca gagaactgaa ttttgttgttggttgtttca ggggatctca tggccgacca 1020 aggacaaggt gcagcaagcc aactactacggctcctcacc caggcctcca ccatcagagt 1080 cggcagctac aacggggagg agatctatgcgccgttcaag agcctccttc ccatggtaat 1140 ctattataga cttgactaat actcttctttttactgaaac caaacataca taacaaagca 1200 tattccgtaa ggtgctagtt gatgttataaaatgaacctg tctttcaggc cagtggtctc 1260 aagtaaacgg aatgttaatc attgggttgaaaaaacaaag gttctaattt tgtgaaagga 1320 aagttaaact tagcataatg aaaaggggaagcactgtaag aaaggtgctg aaacaatcga 1380 ctcggtctgc catgttgtga tcctacttgcaagtcaaaag gttctgtggt tagcccaaag 1440 gttccagcat ctttggatta cactcgtgcagtattgacga tggtgctaac tggttgcaga 1500 ttcgcagact cggtgtttgt tatcttcttttcatgaccaa gtgttaaact ggttttcagg 1560 tgaacccaga cgacattgtg ttcggaggctgggacattag caacatgaac ctggccgact 1620 ccatgaccag ggccaaggtg ctggatattgacctgcagaa gcagctcagg ccctacatgg 1680 agtccatggt gccacttccc cggtatctatgatccggact tcatcgcggc taaccagggc 1740 tctcgcgcca acagtgtcat caagggcaccaagaaagaac aggtggagca gatcatcaag 1800 gatatcaggt atatggatat ggatgctaacgtgccttggt gctaaggtgc acccagtgca 1860 acctaaaaca aataaatact actatgaatttggtaaatat acatacatat cagagcatat 1920 tgtttaaccg gtgcacttag gagtctgcatggtatgttgg acaatttgac attcgatata 1980 cagtgaccgc tcacttgcat gaggactccacaaagaacta aaactactga aagcttaagc 2040 aactattcgt agctaatgat gtatttggtggacatggttt gaagatctag attaacgtgg 2100 ttgaagaaat atggttcact agtataagtaatccattaca gaagcaatgg cttatgtagc 2160 taatgaaaca gggagtttag ggagaagaacaaagtggaca agatagttgt gttgtggact 2220 gcaaacactg aaaggtatag caatgtgtgcgctggtctca acgacacgat ggagaatcta 2280 ctggcatctg tggacaagaa cgaggcggaggtatcaccat caacactata tgccattgcc 2340 tgtgtcatgg agggggtgcc gttcatcaatgggagccccc agaacacctt tgtgcctggt 2400 gcgtggtttg gtgtgtttgc aaaagcctcatggtgttgca tttctgttcc aaagtttcat 2460 ggtgttgtat ttctgttcca aggcttattatacctgttgc atgttcgtag ggctgattga 2520 tcttgctata aaaaacaact gcttgattggtggtgacgac ttcaagagtg gacagaccaa 2580 gatgaaatct gtcttggtcg atttccttgttggtgctgga ataaaggtgg gaacctagta 2640 tctctcttct attaagatga agtgtttttttggcaaatga cgttattgca ataactcttc 2700 tatattttca ttttcatgca gcccacctcaatcgtgagct acaaccactt gggaaacaac 2760 gatggcatga acctgtctgc ccttcaaacattcaggtcca aggagatctc caagagcaac 2820 gtggtggatg acatggtctc gagcaatgccatcctctatg agcccggcga gcatcccgat 2880 catgtcgttg tcatcaaggt ctgttagctgatctttcacc tcgttaaaag ttgacatatg 2940 caaggcagat ttacattgaa acttgtcactcttttgttgc agtatgtgcc gtacgtggga 3000 gacagcaaga gggctatgga cgagtacacctcagagatct tcatgggcgg caagaacacc 3060 atcgtgctgc acaacacctg tgaggactcgctcctcgccg cacctatcat ccttgatctg 3120 gtgctcttgg ctgagctcag caccaggatccagctgaaag ctgagggagg ggtaagagcc 3180 ccccaagtga ttaacctgaa agcacgctgcacgctaggtg atatagcact tttaatacct 3240 tctggtgtct ctcttatgca ggacaaattccactccttcc acccggtggc caccatcctg 3300 agctacctca ccaaggcacc cctggtaagccttttctcct gcatcccggc atcactgcac 3360 tgcgttttgc ttcaatccag ccactgatcgtctctcttga aacctgaaca acaggttccc 3420 cctggcacac cggtggtgaa cgctctggccaagcagacgg cgatgctgga gaacatcatg 3480 agggcctgcg ttgggctggc cccagagaacaacatgatcc tggagtacaa gtgagccaag 3540 tggcgt 3546 <210> SEQ ID NO 15<211> LENGTH: 3546 <212> TYPE: DNA <213> ORGANISM: Zea mays <400>SEQUENCE: 15 ctcgctacct cgcttcgcat tccattcgag gagagcggtg agaggggaggaaaggcaaga 60 tgttcatcga gagcttccgc gtcgagagcc cccacgtgcg gtacggcccgacggagatcg 120 agtcggagta ccggtacgac acgacggagc tggtacacga gggcaaggacggcgcctcac 180 gctgggtcgt ccgccccaag tccgtcaagt acaacttccg gaccagaaccgccgtcccca 240 agctcgggta tgtacggatg cagcggccct agcctcactc tctgtgaaccctcctcctcc 300 cgtgctcagt caaatcctcc gtcgagatca actggtcggc gttccctcctaaatcctaat 360 gaaaatctta ctgctttgcc tgaagacgaa ccgtcgtaat tgttgacagctacgcacaca 420 cttgcccatc cggatgcgtc aaatcagctc gatttgaaat tcgattcgatggtgcccttt 480 tccatatttc gatcatcctt cgcctactgt gcaatgatta cagaaacgtccctttcctct 540 gaactttgtc ttaggctttt tgtcctgtgc acgtgagctg gtatcaatttgttcatgtaa 600 gatcaaattc cagcagggac gatgagcagc agacagaact cattacgctagcaaattgat 660 actaggatta ctggcaagtg tgcatacggc gcaatctgcc atcctggaccccctttgttt 720 aattcctgtt cctatgcatg ttgcctacgt gcagctcgtt gtgtgttatggtgtcaggct 780 gtcagccgct tgtctctgtc tgacggatga tgccaacttt tctgttctggtggtgcaggg 840 tgatgcttgt ggggtgggga ggcaacaacg ggtccacgct gacggctggggtcattgcca 900 gcagggagtg agtagtactt aatttgtcct atattgcttt ccgttgttttcagttattaa 960 tggcctgaca gagaactgaa ttttgttgtt ggctgtttca ggggatctcatggccgacca 1020 aggacaaggt gcagcaagcc aactactacg gctcctcacc caggcctccaccatcagagt 1080 cggcagctac aacggggagg agatctatgc gccgttcaag agcctccttcccatggtaat 1140 ctattataga cttgactaat actcttcttt ttactgaaac caaacatacataacaaagca 1200 tattccgtaa ggtgctagtt gatgttataa agtgaacctg tctttcaggccagtggtctc 1260 aagtaaacgg aatgttaatc attgggttga aaaaacaaag gttctaattttgtgaaagga 1320 atgttaaact tagcataatg aaaaggggaa gcattgtaag aaaggtgctgaaacaatcga 1380 ctcggtctgc catgttgtga tcctacttgc aagtcaaaag gttctgtggttagctcaaag 1440 gttccagcat ctttggatta cactcgtgca gtattgacga tggtgctaactggttgcaga 1500 ttcgcagact cggtgtttgt tatcttcctt tcatgaccaa gtgttgaactggttttcagg 1560 tgaacccaga cgacattgtg ttcggaggct gggacattag caacatgaacctggccgact 1620 ccatgaccag ggccaaggtg ctggatattg acctgcagaa gcagctcaggccctacatgg 1680 agtccatggt gccacttccc cggtatctat gatccggact tcatcgcggctaaccagggc 1740 tctcgcgcca acagtgtcat caagggcacc aagaaagaac aggtggagcagatcatcaag 1800 gatatcaggt atatggatat ggatgctaac gtgccttggt gctaaggtgcacccagtgca 1860 acctaaaaca aataaatact actatgaatt tggtaaatat acatacatatcagaacatat 1920 tgtttaaccg gtgcacttag aagtctgcat ggtatgttgg acaatttgacattcgatata 1980 cagtgaccgc tcacttgcat gaggactcca caaagaacta aaactactgaaagcttaagc 2040 aactattcgt agctaatgat gtatttggtg gacatggttt gaagatctagattaacgtgg 2100 ttgaagaaat atggttcact agcataagta atccattaca gaagctatggcttatgtagc 2160 taatgaaaca gggagtttaa ggagaagaac aaagtggaca agatagttgtgttgtggact 2220 gcaaacactg aaaggtatag caatgtgtgc gctggtctca acgacacgatggagaatcta 2280 ctggcatctg tggacaagaa cgaggcggag gtatcaccat caacactatatgccattgcc 2340 tgtgtcatgg agggggtgcc gttcatcaat gggagccccc agaacacctttgtgcctggt 2400 gcgtggtttg gtgtgtttgc aaaagcttca tggtgttgca tttctgttccaaagtttcat 2460 ggtgttgtat ttccgttcca aggcttatta tacctgttgc atgttcgtagggctgattga 2520 tcttgctata aaaaacaact gcttgattgg tggtgacgac ttcaagagtggacagaccaa 2580 gatgaaatct gtcttggtcg atttccttgt tggtgctgga ataaaggtgggaacctagta 2640 tctctcttct attaagatga agtgtttttt tggcaaatga cgttattgcaataactcttc 2700 tatattttca ttttcatgca gcccacctca atcgtgagct acaaccacttgggaaacaac 2760 gatggcatga acctgtctgc ccttcaaaca ttcaggtcca aggagatctccaagagcaac 2820 gtggtggatg acatggtctc gagcaatgcc atcctctatg agcccggcgagcatcccgat 2880 catgtcgttg tcatcaaggt ctgttagctg atctttcacc tcgttaaaagttgacatatg 2940 caaggcagat ttacattgaa acttgtcact cttttgttgc agtatgtgccgtacgtggga 3000 gacagcaaga gggctatgga cgagtacacc tcagagatct tcatgggcggcaagaacacc 3060 atcgtgctgc acaacacctg tgaggactcg ctcctcgccg cacctatcatccttgatctg 3120 gtgctcttgg ctgagctcag caccaggatc cagctgaaag ctgagggagaggtaagagcc 3180 ccccaagtga ttaacctgaa agcacgctgc acgctaggtg atatagcacttttaatacct 3240 tctggtgtct ctcttatgca ggacaaattc cactccttcc acccggtggccaccatcctg 3300 agctacctca ccaaggcacc cctggtaagc cttttctcct gcatcccggcatcactgcac 3360 tgcgttttgc ttcaatccag ccactgatcg tctctctcga aacctgaacaacaggttccc 3420 cctggcacac cggtggtgaa cgctctggcc aagcagacgg cgatgctggagaacatcatg 3480 agggcctgcg ttgggctggc cccagagaac aacatgatcc tggagtacaagtgagccaag 3540 tggcgt 3546 <210> SEQ ID NO 16 <211> LENGTH: 1070 <212>TYPE: DNA <213> ORGANISM: Zea mays <400> SEQUENCE: 16 cggcacgaggttgcgggcga accgaaaatc acgggcgcga gagatcggag cacggcatgt 60 cggaggagcagttcctcgcc gtggcggtgg aagccgccaa gagcgccggc gagattattc 120 gcaagggattctaccagacc aagaacgtcc agcacaaggg ccaggtggat ttggtgacgg 180 agacggacaaggcctgcgag gacctcatct tcaaccacct ccggaagcac ttcccggacc 240 acaagttcatcggggaggag gagtccgcgg cgctcggggc caccgctgac ctcaccgacg 300 accccacctggatcgtcgat cccctcgacg ggaccactaa tttcgtccat ggtttcccat 360 ttgtatgtgtctccgttggc ctcaccattg ggaaaattcc cactgtcgga gtcgtcttca 420 accccatcatgaacgaactt ttcacggcgg ttcgtggaaa aggggctttc ctgaatggct 480 ctccaattaaagcatcatct caagatgagt tagtgaaggc tcttctggta acagaggctg 540 gaaccaatagagacaagacc actgtggatg atacaaccaa cagaatcaac aggctactat 600 acaagattcgatccatacgg atgtgtggat cattggcttt aaacatgtgt ggagttgcct 660 gtggtagactagatttgtgt tatgagatag gatttggtgg tccatgggat gttgctgctg 720 gtgctgtaattcttcaggaa gccggtggcc ttgtttttga cccaagcggc ggagagtttg 780 atttgatgtcgcgaagaatg gcaggatcaa acagcttgct gaaggataag ttcgtcaagg 840 aactgggggatactaattga aacaaatgtt agtattattc gtggaacaga ttaagacaat 900 aaggttgccccgccgcatgg tgattaactt attgtttggg caacaaaatt ccatgtaatt 960 ctgcacctgtacaactatgt tggacgcaga acattttatt gagttttgtg attacatggg 1020 aatacataggttgaggcaac ggtccctact ttaaaaaaaa aaaaaaaaaa 1070 <210> SEQ ID NO 17<211> LENGTH: 267 <212> TYPE: PRT <213> ORGANISM: Zea mays <400>SEQUENCE: 17 Met Ser Glu Glu Gln Phe Leu Ala Val Ala Val Glu Ala Ala LysSer 1 5 10 15 Ala Gly Glu Ile Ile Arg Lys Gly Phe Tyr Gln Thr Lys AsnVal Gln 20 25 30 His Lys Gly Gln Val Asp Leu Val Thr Glu Thr Asp Lys AlaCys Glu 35 40 45 Asp Leu Ile Phe Asn His Leu Arg Lys His Phe Pro Asp HisLys Phe 50 55 60 Ile Gly Glu Glu Glu Ser Ala Ala Leu Gly Ala Thr Ala AspLeu Thr 65 70 75 80 Asp Asp Pro Thr Trp Ile Val Asp Pro Leu Asp Gly ThrThr Asn Phe 85 90 95 Val His Gly Phe Pro Phe Val Cys Val Ser Val Gly LeuThr Ile Gly 100 105 110 Lys Ile Pro Thr Val Gly Val Val Phe Asn Pro IleMet Asn Glu Leu 115 120 125 Phe Thr Ala Val Arg Gly Lys Gly Ala Phe LeuAsn Gly Ser Pro Ile 130 135 140 Lys Ala Ser Ser Gln Asp Glu Leu Val LysAla Leu Leu Val Thr Glu 145 150 155 160 Ala Gly Thr Asn Arg Asp Lys ThrThr Val Asp Asp Thr Thr Asn Arg 165 170 175 Ile Asn Arg Leu Leu Tyr LysIle Arg Ser Ile Arg Met Cys Gly Ser 180 185 190 Leu Ala Leu Asn Met CysGly Val Ala Cys Gly Arg Leu Asp Leu Cys 195 200 205 Tyr Glu Ile Gly PheGly Gly Pro Trp Asp Val Ala Ala Gly Ala Val 210 215 220 Ile Leu Gln GluAla Gly Gly Leu Val Phe Asp Pro Ser Gly Gly Glu 225 230 235 240 Phe AspLeu Met Ser Arg Arg Met Ala Gly Ser Asn Ser Leu Leu Lys 245 250 255 AspLys Phe Val Lys Glu Leu Gly Asp Thr Asn 260 265 <210> SEQ ID NO 18 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: primer <400> SEQUENCE: 18 acgaggttgcgggcgaaccg aaaat 25 <210> SEQ ID NO 19 <211> LENGTH: 23 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: primer <400> SEQUENCE: 19 tagggaccgt tgcctcaacc tat 23<210> SEQ ID NO 20 <211> LENGTH: 362 <212> TYPE: DNA <213> ORGANISM: Zeamays <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)...(362) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 20ggtaaagggt gcacatttna tggttgggtt gaagggccta tcaccttccc cccaacgtat 60aaatacgagt ttaactcaga aaaatatgta antgacgcga cgaaatctgg gagaagaaca 120cccgcatggt atgctccact agacatcaaa cttgagatat gtctatggaa ataaggaaat 180taacatcttc acctgcttgt ataggtgtga ccgcatcctc tcgtatgggg aggggacaag 240gctactttca tacaacaggg cggagttata tnatctgatc atcgaccggt gactgcagtn 300tatatggcag angttgaaat gtctggcccc atgaagctgc aaagagctct aanattcagc 360 aa362 <210> SEQ ID NO 21 <211> LENGTH: 274 <212> TYPE: DNA <213> ORGANISM:Zea mays <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION:(1)...(274) <223> OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 21ttgngaccat gtngccgtan gcccgacgga gatcgagtcg gagtaccgta nacacgacga 60gctngtgcac gaggccaagg acggcgcctc ccgctggtcg tccgccncaa gtccgtcaat 120acaattccgg accagcagcg ccgtccccaa gctcgggtca tgcttgtggg gttgggaggc 180aacaanggtc cacgctgacg gtggggtcat tggcancagg gagggatctn atgggggaca 240aggacaggtg cgcaagccaa taataaggtn ctna 274 <210> SEQ ID NO 22 <211>LENGTH: 685 <212> TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(685) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 22 cntggaccac gcgtccgcgaaaattgagaa acattgttca gtggacgccg ttctttcaaa 60 cttacaaaaa acagaggtatccatgggtac agctagccgg acaccaaggc aatttcaaag 120 ccggtccgga acctggtacgatcctcaaga aactttgtcc caaagaacag ttgtgcttcc 180 aagtgctgat gaaggacgttctgagaccgt acgtgcccga atacaagggc cacttgacta 240 ccgacgacgg agacctatatcttcagctag aagacttgtt gggtgacttc acttcgccgt 300 gcgtcatgga ctgcaagatcggcgtcagga cgtatctgga agangaactg gcgaaagcca 360 aagagaaacc caagttgagaaaagacatgt acgaaaaaat gattcagata gaccccaacg 420 caccatcgga agangaacaccgactgaagg gtgtgacaaa accgaagtac atggtttgga 480 aggagacnat ttcgtccacngccacgttgg gcttccggat cgaagggatc aanaaaagcn 540 atggaaaatc nagcaaggacttccagacga caaagaaccg ggaccaggtg atcnaacctt 600 tcgagatttc ntccccngtttcccccccgt tatccccaan tncataaacc gactganaac 660 natcaganac ttctggtgaactccn 685 <210> SEQ ID NO 23 <211> LENGTH: 333 <212> TYPE: DNA <213>ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(333) <223> OTHER INFORMATION: n = A,T,C or G <400>SEQUENCE: 23 gctcagagca gattgacttg attctagagg aactttcata tattgatcaagagaagcaag 60 ctagcttcgg tgagatcttt caaggaaaat gtcttaaagc aatgataattggatgtggtt 120 tggtgttctt tcagcaggtc actggtcaac ctagcgttct atactatgctgctacaattt 180 ttcagagtgc tggattctct ggggcatctg atgccactcg tgtgncaattcttcttggct 240 tactgaagct aatcatgacc ggagtagcag tccntgggtc gacagacttggcaggaganc 300 cattgcttat nggaggngtc agtggnatta ctg 333 <210> SEQ ID NO24 <211> LENGTH: 346 <212> TYPE: DNA <213> ORGANISM: Zea mays <220>FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (1)...(346) <223>OTHER INFORMATION: n = A,T,C or G <400> SEQUENCE: 24 gctacctactactcaagtat ccatccttat tgagtacagt gttgatccat ggactcggaa 60 ggagttgtagcagcaaaggt ggcagatgag actactaaac cggcaatcca agaagacggc 120 gccgagagcaaggccgggat gactgatctg ctgatgctga ccgacaagtc gcagctgcag 180 gcgctggcgatgctgctgcg gaacaacgag gagctcatga tgagccaggc gatcaagtcg 240 gagacggaagcgcattgagt acctcaagac ggtgagcgac tgctacacgc ggangatgaa 300 gctcctcgacgattccatgg cggccaggac cacgtacgan cgttcg 346 <210> SEQ ID NO 25 <211>LENGTH: 446 <212> TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE:<221> NAME/KEY: misc_feature <222> LOCATION: (1)...(446) <223> OTHERINFORMATION: n = A,T,C or G <400> SEQUENCE: 25 gcctccgnng cttcttccctccctcaaatc aggaaaccat ccaacttgtc gatcggcctt 60 gcttgccttg gtactcctgcttccgccatg gttcagatca aagagttgta tcctccccca 120 ttccaagttc ttgtgtcagcagtgtttaat tctggtaccc gcgtttgaat tttgctgtat 180 atattatttg cgcgtataccttgactcgaa tctcgcgcga cgtacgaaag ccggatcgtc 240 atgcccatgt ccatggaagagtacgagata gggctgagct acaccatcat gaagatggag 300 cagcagaaca ccaacagcaaggagggcgtg gaggtgctgc agcaggcccc gttccacgag 360 gatgccaagc ttggcaagggccacttcact tccaaagttt atcatctgca aagcaagatt 420 ccgtcatgga tgaagggctttcacct 446 <210> SEQ ID NO 26 <211> LENGTH: 549 <212> TYPE: DNA <213>ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(549) <223> OTHER INFORMATION: n = A,T,C or G <400>SEQUENCE: 26 ggcacgagca aggtggttgc acaccagagc tctgcttctc ggatgatgattttgatatgg 60 tacctgattg ccaccggaaa cctctgatta gacttggtgc acacatgccagcccgggcag 120 agcaagcatc caggaggagt gaattcgacc cgcttctcct aacaggcggtggattcctgt 180 tcccaaacca gaccggcgaa tgcatgatgt gatcctattc tttgggataatcgacatcct 240 ccaggattac agcttaagaa agcgggccga gcatgcttac aagtcattacagacagatcc 300 caactcgatc tctgccgtgg acccgaagct ctactcgaag agtttccaagacttccatcg 360 ggcagaattt ttgtggaaat ggctaanngg cntggatagn nttaaccgcgaattccatgg 420 cggaggccag agcacntgtn aaggattccg tgggcatttt tttgcgcgcatnaagaatct 480 anctaatgcc agaatcatct tcatccnggg gatcngtaaa cagcaccggtgggaactact 540 gtgaagcnt 549 <210> SEQ ID NO 27 <211> LENGTH: 434 <212>TYPE: DNA <213> ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY:misc_feature <222> LOCATION: (1)...(434) <223> OTHER INFORMATION: n =A,T,C or G <400> SEQUENCE: 27 ccacgcgtcc gcggnacgct ggcgtnccaaatggggaagt atttgacccc aaggctagtt 60 tgccagtgaa gaaaaccctc aaggtgaaagtatatatggg agacgggtgg ngccatggac 120 ttcagtaaaa ctcatttcga tgccttttcgcctccagatt tctatactag ggtagggatc 180 gcaggtgtga aggcagacag tgtgatgaagaagacaaggg tgattgagga ccagtgggtg 240 ccgatgtggg atgaggagtt cacgttccttctgaacggtt ccggagctgg ccctcctgag 300 ggtaagaagg tccaaggaat acgaacatgtcggagaagca cgancttccg ggggggcaga 360 ncagtgttgc cggtattggg agctgaagcagggcatccgt gcctgtgccc ctgcacgatc 420 gcaagggtgt aagg 434 <210> SEQ ID NO28 <211> LENGTH: 1082 <212> TYPE: DNA <213> ORGANISM: Zea mays <400>SEQUENCE: 28 agaattcggc acgaggttgc gggcgaaccg aaaatcacgg gcgcgagagatcggagcacg 60 gcatgtcgga ggagcagttc ctcgccgtgg cggtggaagc cgccaagagcgccggcgaga 120 ttattcgcaa gggattctac cagaccaaga acgtcgagca caagggccaggtggatttgg 180 tgacggagac ggacaaggcc tgcgaggacc tcatcttcaa ccacctccggaagcacttcc 240 cggaccacaa gttcatcggg gaggaggagt ccgcggcgct cggggccaccgctgacctca 300 ccgacgaccc cacctggatc gtcgatcccc tcgacgggac cactaatttcgtccatggtt 360 tcccatttgt atgtgtctcc gttggcctca ccattgggaa aattcccactgtcggagtcg 420 tcttcaaccc catcatgaac gaacttttca cggcggttcg tggaaaaggggctttcctga 480 atggctctcc aattaaagca tcatctcaag atgagttagt gaaggctcttctggtaacag 540 aggctggaac caatagagac aagaccactg tggatgatac aaccaacagaatcaacaggc 600 tactatacaa gattcgatcc atacggatgt gtggatcatt ggctttaaacatgtgtggag 660 ttgcctgtgg tagactagat ttgtgttatg agataggatt tggtggtccatgggatgttg 720 ctgctggtgc tgtaattctt caggaagccg gtggccttgt ttttgacccaagcggcggag 780 agtttgattt gatgtcgcga agaatggcag gatcaaacag cttgctgaaggataagttcg 840 tcaaggaact gggggatact aattgaaaca aatgttagta ttattcgtggaacagattaa 900 gacaataagg ttgccccgcc gcatggtgat taacttattg tttgggcaacaaaattccat 960 gtaattctgc acctgtacaa ctatgttgga cgcagaacat tttattgagttttgtgatta 1020 catgggtata cataggttga ggcaacggtc cctactttaa aaaaaaaaaaaaaaaactcg 1080 ag 1082 <210> SEQ ID NO 29 <211> LENGTH: 1330 <212>TYPE: DNA <213> ORGANISM: Zea mays <400> SEQUENCE: 29 aggtgacactattagaagag ctatgacgtc gcatgcacgc gtacgtaagc ttggatcctc 60 tagagcggccgccctttttt tttttttttt ttttagagga gtcgtaaagg aattttatag 120 gaatcagtttattttcacag gaaatacata ggaaatggga aaaaatccca catttcaaag 180 aaggcctaaattggatccaa acagttggcc ttaatcaatt tgctcggcca ggtagaatag 240 tacctggtaaaggtaccaag catgccctaa cccttctgtc aatcagtatt cgccatagtt 300 caatcaatttgaaacggtgt ctccacttgg ctgctggcca cattgccggt tttgctatat 360 atatatgcccaaggcaaatc gtttctgaaa aactgataca ggaagaattc tcgcatacaa 420 actacgagcatatacacagc agaacttctg gctgctcatt caagattcag cgttgggaat 480 cttcatcgggatgtgtactg tagagtgagt tcactttrgc ayttttgwac atgttgtcaa 540 ttcacgcccttggtwgtact ttgagcagtt gcgggacgyt tcttttttgt atgtcgggkt 600 ratcaaaatcacggtccatg tcaaaacggt actggttcca rgtgcamatc ctttacgaat 660 tcaaaacctttgagccttyt tctcttttcc tattcttgac agctctccta aaatgtattc 720 cttttgttcytggattawtg cacaaggact cgaaaatcac macttawtcc atttgctgca 780 gcccaactyctcgagaacct ccttgtttgg gattgaccac agcaacaaga aaggactcaa 840 agctgttcccatatatccat atcgagtcta tagcagaaac aagaccataa acattctcca 900 aattttcaactgccacatat tcaccctgtg aaagtttgaa tatattcttt ttacggtcta 960 tgattttcatagatccatca ggttgccact caccaatgtc accagtgtgg aaccatccat 1020 caatgaggacctcctttgta aggtcttcac gcttgtagta tcctgagaat aatgtttctc 1080 ccctgatgcatatctctcca cgaggtttgc tagcaagtgc atcataatcc atttctggga 1140 ccgactccagacgaacatcg atgtttggca ctggggggcc aacagttcct atcatggaca 1200 tttgatttggtagcgagacg aaagatccag cacaagtttc cgcggacgcg tgggtcgacc 1260 cgggaattccggaccggtac ctgcaggcgt accagctttc cctatagtga gtcgtattag 1320 agctttggcg1330 <210> SEQ ID NO 30 <211> LENGTH: 720 <212> TYPE: DNA <213>ORGANISM: Zea mays <220> FEATURE: <221> NAME/KEY: misc_feature <222>LOCATION: (1)...(720) <223> OTHER INFORMATION: n = A,T,C or G <400>SEQUENCE: 30 gcagacggaa tgaagacttt gaccatgtct ttagatcaat gacattttcttctccttcaa 60 atggattatt gacaacatca gtttctggtt ctgctgctca gcttcttcgaggaacaaatg 120 gatcaagact gcctgagttg tcagatactg acttgatcgt gtttcttggtgacttcaatt 180 accggcttta taacatttct ttcgatgagg caatgggctt ggtttcccggcgatgctttg 240 actggttgag agagaatgat cagctgcgag cagaaatgaa atctgggagagtcttccagg 300 gattacgtga aggagaattt aagttccccc ctacttataa atttgagaagcacatagcan 360 gcttatctgg ttatgataat agtgagaaaa ggcgcattcc aagcctggtgtgacagagtt 420 ctatatcgag acagccgaac tagttcacag attgagtgtt ctttggaatgtcctgtagtc 480 tgttcgatat cactgtacga ctcttgtatg gaagcaacag acagtgatcataaacctgtc 540 aaatgtgtgt tcaatttaga tattgctcat gtggacaaaa caaaacaatgangcaaaant 600 atggagaaat aatgggttca aataaaggaa tgcntgactc acttcaaggccttggaggct 660 ttgcctgaan tagatatcaa cacgaatgac atcantccgc aagatnaaaanccntttgtg 720 <210> SEQ ID NO 31 <211> LENGTH: 1255 <212> TYPE: DNA<213> ORGANISM: Zea mays <400> SEQUENCE: 31 ccgggtcgac ccacgcgtccgcggacgcgt gggcccttta cacgctcgat tccccccgcc 60 gtttccacgg gggccaggtgcctgcattac tgggtccggg cctagcgtca gcgcaaccga 120 acacgccggc gaccgctgccgcccttctcc cagctgccca ggtcttcgtc gggccctttg 180 cctgcggcgt cggcgacgagcgcctgccac accagggtat tttaggatca tcataaattt 240 cattgcattt gcggtttgctattccaacaa ggctctcttt gctatcctgt acttcatcag 300 ctttgtcctt gatggtgtggatggttggtt tgcaaggaag ttcaatcaag catcaacctt 360 tggagctgtg ttagacatggttacagatag ggttagcact gcttgtttgt tggcccttct 420 ctcccagttt tacagacytggtttagtctt tcttgatatt gctttggatt ggwtattacg 480 agccactggt ttcaaatkkacmagttcttt tctttgtcag gtargactta gccacaaggw 540 tgtaaaacac acaggcaattggcttctgaa attatattat gggtacaggc cattcatgsc 600 cttctgctgt gttycttgtraggtttwata tawtttccyg tttctctttg ctgawgarga 660 gtcaacaarc cttgcttagtgtatgcaaaa ggmatcctga accaaartcc cgttcgttaw 720 cytggkgttk gtttcmacycwagttggctg ggcagtgaag caagccacca acgtcatcca 780 gatgaaaact gctgcggacgcatgcgtggt gtatgatctg aagcgcagca aatgaagcat 840 gaaggcagct tcacggtttagtatcgacat atccaaggga aaactctgcg agggggcggg 900 ctacgtcttg cgtgccttgacatctttctg atgatgcggt catatgtggg accaggggat 960 gacatgccgt ggccaatgcaaacaattgtt ttgtgaaagc agcggccgtt aagttgttgt 1020 cagtgtgaga gtggtgatgcgatcatgatc ctttttacct agagtagctc ccctttgtgt 1080 tagcctgaac gatgttttgcaagccgcatg ttccgaactc taggattatt tggattacaa 1140 aacttacata ttccatcctcaaaaaaaaaa aaaaagggcg gccgctctag aggatccaag 1200 cttacgtacg cgtgcatgcgacgtcatagc tcttctatag tgtcacctaa attca 1255

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence which has at least 65% sequence identity to SEQ ID NO: 6,wherein the % sequence identity is based on the entire sequence and isdetermined by the GAP program where the gap creation penalty=12 and thegap extension penalty=4.
 2. An isolated polypeptide comprising thesequence of SEQ ID NO: 6.